Humanized collagen antibodies and related methods

ABSTRACT

The invention provides a grafted antibody, or functional fragment thereof, comprising one or more complementarity determining regions (CDRs) having at least one amino acid substitution in one or more CDRs of a heavy chain CDR, where the grafted antibody or functional fragment thereof has specific binding activity for a cryptic collagen epitope. The invention also provides methods of using an antibody having specific binding activity for a cryptic collagen epitope, including methods of inhibiting angiogenesis, tumor growth, and metastasis.

BACKGROUND OF THE INVENTION

[0001] The present invention relates generally to immunology and morespecifically to humanized antibodies and uses thereof.

[0002] The extracellular matrix (ECM) plays a fundamental role in theregulation of normal and pathological processes. The most abundantlyexpressed component found in the ECM is collagen. Triple helicalcollagen is known to be highly resistant to proteolytic cleavage exceptby members of the matrix metalloproteinase (MMP) family of enzymes.

[0003] Angiogenesis and tumor growth depend on cellular interactionswith the extracellular matrix. During angiogenesis and tumor invasion,both endothelial cells as well as tumor cells proteolytically remodeltheir extracellular microenvironment. The invasive cells then interactwith this newly remodeled extracellular matrix followed by migration andinvasion. To this end, a major component of the basement membranesurrounding blood vessels is collagen-IV. Moreover, collagen-I is themajor component of the interestitial matrix.

[0004] One of the major detrimental consequences of the progression ofcancer is metastasis beyond the site of the primary tumor. Suchmetastasis often requires more aggressive therapies, and once metastasishas occurred, the prognosis for survival of a cancer patient decreasesdramatically.

[0005] The growth of all solid tumors requires new blood vessel growthfor continued expansion of the tumors, particularly beyond a minimalsize. Because angiogenesis is required for tumor growth, inhibitingangiogenesis is one approach to inhibiting tumor growth. It is thereforedesirable to identify molecules that can target angiogenic vasculature.Particularly attractive molecules for targeting angiogenic vasculatureare antibodies that can bind specifically to angiogenic vasculature.However, since most antibodies are developed in non-human animals suchas mice, these antibodies often have undesirable immunogenic activitythat limits their effectiveness for human therapy.

[0006] One approach to overcoming the detrimental properties ofnon-human antibodies is to humanize the antibodies by using humanantibody framework region sequences spliced together with the bindingdomains that confer binding specificity. However, grafting of thesebinding domains, referred to as complementarity determining regions(CDRs), into human frameworks has often resulted in the loss of bindingaffinity.

[0007] Thus, there exists a need to identify antibodies specific forangiogenic vasculature and to humanize and optimize the antibodies fortherapeutic purposes. The following invention satisfies this need andprovides related advantages as well.

SUMMARY OF THE INVENTION

[0008] The invention provides a grafted antibody, or functional fragmentthereof, comprising one or more complementarity determining regions(CDRs) having at least one amino acid substitution in one or more CDRsof a heavy chain CDR, where the grafted antibody or functional fragmentthereof has specific binding activity for a cryptic collagen epitope.The invention also provides methods of using an antibody having specificbinding activity for a cryptic collagen epitope, including methods ofinhibiting angiogenesis, tumor growth, and metastasis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 shows the sequences of primers used to clone nucleic acidsencoding HUIV26 and HUI77 antibodies. FIG. 1A shows a set of 5′ primersfor the signal peptide of mouse antibody light chain (SEQ ID NOS:184-192). FIG. 1B shows a set of 5′ primers for the signal peptide ofmouse antibody heavy chain (SEQ ID NOS: 193-211). FIG. 1C shows a set ofprimers for the constant region of mouse heavy and light chains. Primer2650 (SEQ ID NO:212) is the 3′ primer for mouse kappa light chainconstant region (amino acids 123-115). Primer 2656 (SEQ ID NO:213) isthe 3′ primer for mouse IgM CH1 region (amino acids 121-114). Primer2706 (SEQ ID NO:214) is the 3′ primer for mouse IgM CH1 region (aminoacids 131-124).

[0010]FIG. 2 shows the sequence of the variable region of anti-crypticcollagen site antibody HUIV26. FIG. 2A shows the nucleotide sequence ofHUIV26 variable region light chain (SEQ ID NO:1). FIG. 2B shows thenucleotide sequence of HUIV26 variable region heavy chain (SEQ ID NO:3).FIG. 2C shows an alignment of the amino acid sequence of HUIV26 lightchain (VK) domain of HUIV26 (SEQ ID NO:2) with a human variable regionfusion, VKIV/JK2 (SEQ ID NO:6) and an alignment of HUIV26 heavy chain(V_(H)) domain (SEQ ID NO:4) with a human variable region fusionVHIII/JH6 (SEQ ID NO:8), with CDRs underlined. Amino acids in theframework region that differ between the aligned sequences are indicatedby lines.

[0011]FIG. 3 shows the sequence of the variable region of anti-crypticcollagen site antibody HUI77. FIG. 3A shows the nucleotide sequence ofHUI77 variable region light chain (SEQ ID NO:9). FIG. 3B shows thenucleotide sequence of HUI77 variable region heavy chain (SEQ ID NO:11).FIG. 3C shows an alignment of the amino acid sequence of HUI77 lightchain (V_(K)) domain of HUI77 (SEQ ID NO:10) with a human variableregion fusion, VKII/JK1 (SEQ ID NO:14) and an alignment of HUI77 heavychain (V_(H)) domain (SEQ ID NO:12) with a human variable region fusionVHIII/JHG (SEQ ID NO:16), with CDRs underlined. Amino acids in theframwork region that differ between the aligned sequences are indicatedby lines. FIG. 3D shows an alignment of the nucleotide sequence of HUI77variable region with the sequence of the human framework fusion of DPK13and JK1 (SEQ ID NO:17).

[0012]FIG. 4 shows beneficial CDR mutations for anti-cryptic collagensite antibody HUIV26. FIG. 4A shows a set of primers used to generaterandom mutations in LCDR3 and HCDR3 of HUIV26 (HUIV26 LCDR3 primers, SEQID NOS:224-232; HUIV26 HCDR3 primers, SEQ ID NOS:233-243). FIG. 4B showsa set of primers used to generate random mutations in LCDR1a (SEQ IDNOS:266-273), LCDR1b (SEQ ID NOS:274-282), LCDR2 (SEQ ID NOS:283-289),HCDR1 (SEQ ID NOS:290-294), HCDR2a (SEQ ID NOS:295-303) and HCDR2b (SEQID NOS:304-311) of HUIV26. FIG. 4C shows beneficial CDR mutations of theHUIV26 antibody.

[0013]FIG. 5 shows beneficial CDR mutations for anti-cryptic collagensite antibody HUI77. FIG. 5A shows a set of primers used to generaterandom mutations in LCDR3 and HCDR3 of HUI77. FIG. 5B shows a set ofprimers used to generate random mutations in LCDR1a (SEQ IDNOS:312-319), LCDR1b (SEQ ID NOS:320-327), LCDR2 (SEQ ID NOS:328-334),HCDR1 (SEQ ID NOS:335-341), HCDR2a (SEQ ID NOS:342-349) and HCDR2b (SEQID NOS:350-357) of HUI77. FIG. 5C shows beneficial CDR mutations of theHUI77 antibody.

[0014]FIG. 6 shows mutations in combinatorial variants of the HUIV26antibody. The position of amino acids are shown, with mutationsdifferent than wild type shown in bold. The relative activity ofcombinatorial variants is shown as “SPEKon” and “SPEKoff” (last column)Primers used to create the combinatorial libraries are also shown (SEQID NOS:163-173).

[0015]FIG. 7 shows mutations in combinatorial variants of the HUI77antibody. The position of amino acids are shown, with mutationsdifferent than wild type shown in bold. The relative activity ofcombinatorial variants is shown as “SPEK_(on”) and “SPEK_(off)” (lastcolumn) Primers used to create the combinatorial libraries are alsoshown (SEQ ID NOS:174-183).

[0016]FIG. 8 shows the activity and specificity of HUIV26 variants. Thebinding of purified Fabs of IX-IV26, containing wild type HUIV26 CDRs,and the HUIV26 variants 2D4H1-C3 and DhuG5 is shown for denaturedcollagen IV (FIG. 8A), denatured collagen I (FIG. 8B) and nativecollagen IV (FIG. 8C).

[0017]FIG. 9 shows the activity and specificity of HUI77 variants. Thebinding of purified Fabs of IX-177, containing wild type HUI77 CDRs, andHUI77 variants Qh2b-B7 and QhuD9 is shown for denatured collagen I (FIG.9A), denatured collagen IV (FIG. 9B) and native collagen I (FIG. 9C).

[0018]FIG. 10 shows the binding activity of the HUIV26 variant DhuH8.The binding activity of the Fab form and the IgG form of the antibody todenatured (d-Iv) and native (n-IV) human collagen IV is shown.

[0019]FIG. 11 shows the effect of the HUI77 variant QH2b on B16 melanomacell proliferation. B16 melanoma cells grown in culture were not treated(control; squares) or treated with the IgG form of the QH2b variant(circles).

DETAILED DESCRIPTION OF THE INVENTION

[0020] The invention provides antibodies specific for a cryptic collagensite, which is exposed during angiogenesis and tumor cell invasionthrough collagenous tissue and thus serves as an antibody that cantarget angiogenic vasculature. The antibodies are optimized for bindingactivity to a cryptic collagen site. The antibodies can be used totarget angiogenic vasculature for diagnostic or therapeutic purposes.The antibodies can also be used to inhibit tumor growth.

[0021] As used herein, the term “CDR” or “complementarity determiningregion” is intended to mean the non-contiguous antigen combining sitesfound within the variable region of both heavy and light chainpolypeptides. These particular regions have been described by Kabat etal., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. ofHealth and Human Services, “Sequences of proteins of immunologicalinterest” (1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987);and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where thedefinitions include overlapping or subsets of amino acid residues whencompared against each other. Nevertheless, application of eitherdefinition to refer to a CDR of an antibody or grafted antibodies orvariants thereof is intended to be within the scope of the term asdefined and used herein. The amino acid residues which encompass theCDRs as defined by each of the above cited references are set forthbelow in Table 1 as a comparison. TABLE 1 CDR Definitions Kabat¹Chothia² MacCallum³ V_(H) CDR1 31-35 26-32 30-35 V_(H) CDR2 50-65 53-5547-58 V_(H) CDR3  95-102  96-101  93-101 V_(L) CDR1 24-34 26-32 30-36V_(L) CDR2 50-56 50-52 46-55 V_(L) CDR3 89-97 91-96 89-96

[0022] As used herein, the term “framework” when used in reference to anantibody variable region is intended to mean all amino acid residuesoutside the CDR regions within the variable region of an antibody. Avariable region framework is generally between about 100-120 amino acidsin length but is intended to reference only those amino acids outside ofthe CDRs. As used herein, the term “framework region” is intended tomean each domain of the framework that is separated by the CDRs.

[0023] As used herein,the term “donor” is intended to mean a parentantibody molecule or fragment thereof from which a portion is derivedfrom, given to or contributes to another antibody molecule or fragmentthereof so as to confer either a structural or functional characteristicof the parent molecule onto the receiving molecule. For the specificexample of CDR grafting, the parent molecule from which the grafted CDRsare derived is a donor molecule. The donor CDRs confer binding affinityof the parent molecule onto the receiving molecule. The donor moleculecan be a different species or the same species as the receivingmolecule. If the donor and receiving molecules are of the same species,it is understood that it is sufficient that the donor is a separate anddistinct molecule from the receiving molecule.

[0024] As used herein, the term “acceptor” is intended to mean anantibody molecule or fragment thereof which is to receive the donatedportion from the parent or donor antibody molecule or fragment thereof.An acceptor antibody molecule or fragment thereof is therefore impartedwith the structural or functional characteristic of the donated portionof the parent molecule. For the specific example of CDR grafting, anacceptor molecule, including framework and/or other antibody fragments,is the receiving molecule into which the CDRs are grafted. The acceptorantibody molecule or fragment is imparted with the binding affinity ofthe donor CDRs or parent molecule. As with a donor molecule, it isunderstood that an acceptor molecule can be the same or a differentspecies as the donor.

[0025] A “variable region” when used in reference to an antibody or aheavy or light chain thereof is intended to mean the amino terminalportion of an antibody which confers antigen binding onto the moleculeand which is not the constant region. The term is intended to includefunctional fragments thereof which maintain some of all of the bindingfunction of the whole variable region. Therefore, the term “heteromericvariable region binding fragments” is intended to mean at least oneheavy chain variable region and at least one light chain variableregions or functional fragments thereof assembled into a heteromericcomplex. Heteromeric variable region binding fragments include, forexample, functional fragments such as Fab, F(ab)₂, Fv, single chain Fv(scFv) and the like. Such functional fragments are well known to thoseskilled in the art. Accordingly, the use of these terms in describingfunctional fragments of a heteromeric variable region is intended tocorrespond to the definitions well known to those skilled in the art.Such terms are described in, for example, Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1989);Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers,R. A. (ed.), New York: VCH Publisher, Inc.); Huston et al., CellBiophysics, 22:189-224 (1993); Plückthun and Skerra, Meth. Enzymol.,178:497-515 (1989); and in Day, E. D., Advanced Immunochemistry, SecondEd., Wiley-Liss, Inc., New York, N.Y. (1990).

[0026] As used herein, the term “population” is intended to refer to agroup of two or more different molecules. A population can be as largeas the number of individual molecules currently available to the user orable to be made by one skilled in the art. Populations can be as smallas 2-4 molecules or as large as 10¹³ molecules. Generally, a populationwill contain two or more, three or more, five or more, nine or more, tenor more, twelve or more, fifteen or more, or twenty or more differentmolecules. A population can also contain tens or hundreds of differentmolecules or even thousands of different molecules. For example, apopulation can contain about 20 to about 100,000 different molecules ormore, for example about 25 or more, 30 or more, 40 or more, 50 or more,75 or more, 100 or more, 150 or more, 200 or more, 300 or more, 500 ormore, or 1000 or more different molecules, and can contain 10,000,100,000 or even 1×10⁶ or more different molecules. Those skilled in theart will know what size and diversity of a population is suitable for aparticular application.

[0027] As used herein, the term “altered” when used in reference to anantibody variable region is intended to mean a heavy or light chainvariable region that contains one or more amino acid changes in aframework region, a CDR or both compared to the parent amino acidsequence at the same position. Where an altered variable region isderived from or composed of donor and acceptor regions, the changedamino acid residues within the altered species are to be compared totheir respective amino acid positions within the parent donor andacceptor regions.

[0028] As used herein, the term “nucleic acid” or “nucleic acids” isintended to mean a single- or double-stranded DNA or RNA molecule. Anucleic acid molecule of the invention can be of linear, circular orbranched configuration, and can represent either the sense or antisensestrand, or both, of a nucleic acid molecule. The term also is intendedto include nucleic acid molecules of both synthetic and natural origin.A nucleic acid molecule of natural origin can be derived from anyanimal, such as a human, non-human primate, mouse, rat, rabbit, bovine,porcine, ovine, canine, feline, or amphibian, or from a lower eukaryote,such as Drosophila, C. elegans, yeast, and the like. A synthetic nucleicacid includes, for example, chemical and enzymatic synthesis. The term“nucleic acid” or “nucleic acids” is similarly intended to includeanalogues of natural nucleotides which have similar functionalproperties as the referenced nucleic acid and which can be utilized in amanner similar to naturally occurring nucleotides and nucleosides.

[0029] As used herein, the term “antibody” is used in its broadest senseto include polyclonal and monoclonal antibodies, as well as antigenbinding fragments of such antibodies. An antibody useful in theinvention, or antigen binding fragment of such an antibody, ischaracterized by having specific binding activity for a polypeptide or apeptide portion thereof of at least about 1×10⁵ M⁻¹. Thus, Fab, F(ab′)₂,Fd, Fv, single chain Fv (scFv) fragments of an antibody and the like,which retain specific binding activity for a polypeptide, are includedwithin the definition of an antibody. Specific binding activity of anantibody for a polypeptide can be readily determined by one skilled inthe art, for example, by comparing the binding activity of an antibodyto a particular polypeptide versus a control polypeptide that is not theparticular polypeptide. Methods of preparing polyclonal or monoclonalantibodies are well known to those skilled in the art (see, for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press (1988)).

[0030] In addition, the term “antibody” as used herein includesnaturally occurring antibodies as well as non-naturally occurringantibodies, including, for example, single chain antibodies, chimeric,bifunctional and humanized antibodies, as well as antigen-bindingfragments thereof. Such non-naturally occurring antibodies can beconstructed using solid phase peptide synthesis, can be producedrecombinantly or can be obtained, for example, by screeningcombinatorial libraries consisting of variable heavy chains and variablelight chains as described by Huse et al. (Science 246:1275-1281 (1989)).These and other methods of making functional antibodies are well knownto those skilled in the art (Winter and Harris, Immunol. Today14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow andLane, supra, 1988); Hilyard et al., Protein Engineering: A practicalapproach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed.(Oxford University Press 1995)).

[0031] As used herein, specific binding means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity, for example, an antibodythat binds a distinct epitope or antigen. Specificity of binding alsocan be determined, for example, by competition with a control molecule,for example, competition with an excess of the same molecule. In thiscase, specific binding is indicated if the binding of a molecule iscompetitively inhibited by itself. Thus, specific binding between anantibody and antigen is measurably different from a non-specificinteraction and occurs via the antigen binding site of the antibody.

[0032] As used herein, selective binding refers to a binding interactionthat is both specific and discriminating between molecules, for example,an antibody that binds to a single molecule or closely relatedmolecules. For example, an antibody can exhibit specificity for anantigen that can be both specific and selective for the antigen if theepitope is unique to a molecule. Thus, a molecule having selectivebinding can differentiate between molecules, as exemplified by anantibody having specificity for an epitope unique to one molecule orclosely related molecules. Alternatively, an antibody can havespecificity for an epitope that is common to many molecules, forexample, a carbohydrate that is expressed on a number of molecules. Suchan antibody has specific binding but is not selective for one moleculeor closely related molecules.

[0033] As used herein the term “binding affinity” is intended to meanthe strength of a binding interaction and includes both the actualbinding affinity as well as the apparent binding affinity. The actualbinding affinity is a ratio of the association rate over thedisassociation rate. Therefore, conferring or optimizing bindingaffinity includes altering either or both of these components to achievethe desired level of binding affinity. The apparent affinity caninclude, for example, the avidity of the interaction. For example, abivalent heteromeric variable region binding fragment can exhibitaltered or optimized binding affinity due to its valency.

[0034] As used herein, the term “substantially the same” when used inreference to binding affinity is intended to mean similar or identicalbinding affinities where one molecule has a binding affinity that issimilar to another molecule within the experimental variability of theaffinity measurement. The experimental variability of the bindingaffinity measurement is dependent upon the specific assay used and isknown to those skilled in the art.

[0035] As used herein, the term “optimizing” when used in reference to avariable region or a functional fragment thereof is intended to meanthat the functional activity of the variable region has been modifiedcompared to the activity of a parent variable region or a donor variableregion, resulting in a desirable change in activity. A variable regionor functional fragment thereof exhibiting optimized activity canexhibit, for example, higher affinity or lower affinity binding, orincreased or decreased association or dissociation rates compared to anunaltered variable region. A variable region or functional fragmentthereof exhibiting optimized activity also can exhibit increasedstability such as increased half-life in a particular organism. Forexample, an antibody activity can be optimized to increase stability bydecreasing susceptibility to proteolysis. An antibody exhibitingoptimized activity also can exhibit lower affinity binding, includingdecreased association rates or increased dissociation rates, if desired.An optimized variable region exhibiting lower affinity binding isuseful, for example, for penetrating a solid tumor. In contrast to ahigher affinity variable region, which would bind to the peripheralregions of the tumor but would be unable to penetrate to the innerregions of the tumor due to its high affinity, a lower affinity variableregion would be advantageous for penetrating the inner regions of thetumor. As with optimization of binding affinities above, optimization ofa catalytic variable region can be, for example, increased or decreasedcatalytic rates, disassociation constants or association constants.

[0036] As used herein, a “cryptic collagen site” or “cryptic collagenepitope” refers to an epitope of a collagen molecule that is lessaccessible to binding of an antibody, or functional fragment thereof, innative collagen than in denatured collagen. An antibody having bindingactivity for a cryptic collagen epitope preferentially recognizesdenatured collagen over native collagen, that is, has a higher bindingaffinity for denatured over native collagen. For example, such anantibody can have at least about a 2-fold or greater preference, thatis, at least about 2-fold higher binding activity, for denatured collageover native collagen, and can exhibit about a 3-fold or greaterpreference, about a 5-fold or greater preference, about a 10-fold orgreater preference, about a 15-fold or greater preference, about a20-fold or greater preference, about a 25-fold or greater preference,about a 50-fold or greater preference, about a 100-fold or greaterpreference, or even a higher preference for denatured over nativecollagen.

[0037] Native collagen herein refers to a molecule where threealpha-chains are organized in a triple helical molecule. Native collagencan be of different stages of post-translational processing such aspro-collagen and any intermediates in the generation of a mature tissueform of collagen, or collagen molecules isolated by limited proteolysisof tissues under conditions where the triple-helical structure ofcollagen is not disrupted. Thus, native collagen can be an intactcollagen molecule or can contain non-triple-helical sequences flankingtriple-helical regions, so long as the triple-helical is not disrupted.Denatured collagen herein refers to collagen where the triple helix iscompletely or partially disrupted such that a cryptic epitope is madeaccessible. Denaturation of collagen can occur in situ by the action ofproteinases, for example, matrix metalloproteinases, that cleavecollagen within triple helical regions, rendering the resultingfragments of the triple helix unstable. Denaturation of collagen can beinduced in vitro by thermal or chemical denaturation of native collagen.Denatured collagen can also be prepared in vitro by treatment of nativecollagen with proteinases capable of cleaving a triple helicalregion(s), which are commonly referred to as collagenolytic enzymes, attemperatures where the resulting fragments of the triple helix arethermally unstable. Denatured collagen can be obtained by denaturationof native collagens of different stages of post-translational processingor denaturation of native collagen isolated from tissues by limitedproteolysis. One skilled in the art will know a variety of methods forisolation of native collagens and a variety of methods to denature atriple helix that contains a cryptic collagen epitope.

[0038] An antibody of the invention can have binding activity for acryptic collagen epitope that is the same as the respective parentalmouse antibody. For example, an antibody of the invention having CDRsderived from HUIV26 can have essentially the same binding specificity asthe mouse HUIV26 antibody described by Xu et al., Hybridoma 19:375-385(2000); Xu et al., J. Cell Biol. 154:1069-1079 (2001); and WO 00/40597,each of which is incorporated herein by reference. Similarly, anantibody of the invention having CDRs derived from HUI77 can haveessentially the same binding specificity as the mouse HUI77 antibodydescribed by Xu et al., supra, 2000; Xu et al., supra, 2001; and WO00/40597. Such binding specificity can be tested by the methodsdisclosed herein, for example, by comparing the activity of an antibodyof the invention to the corresponding parental mouse antibody. Forexample, an antibody of the invention derived from HUIV26 can becompared to a corresponding mouse antibody having the variable regionamino acid sequence shown in FIG. 2C (SEQ ID NOS:2 and 4). Similarly, anantibody of the invention derived from HUI77 can be compared to acorresponding mouse antibody having the variable region amino acidsequence shown in FIG. 3C (SEQ ID NOS:10 and 12). Similar bindingspecificity can be determined, for example, by competitive binding withthe corresponding parental antibody. It is understood that an antibodyof the invention can have essentially the same specificity as thecorresponding parental antibody or can have altered specificity so longas the antibody has binding activity for a cryptic collagen epitope.

[0039] The invention provides antibodies having specific bindingactivity for a cryptic collagen epitope. The antibodies contain at leastone CDR having at least one amino acid substitution in a CDR of theantibodies HUIV26 and HUI77, which are antibodies that bind to a crypticcollagen site. The invention also provides nucleic acids encoding theseantibodies. The invention further provides methods using the antibodies.

[0040] Highly specific monoclonal antibodies have been developed thatrecognize a cryptic domain of human collagen, designated HUIV26 andHUI77 (see Xu et al., Hybridoma 19:375-385 (2000); Xu et al., J. CellBiol. 154:1069-1079 (2001); WO 00/40597, each of which is incorporatedherein by reference). Monoclonal antibody HUIV26 recognizes a crypticdomain of human collagen-IV, and HUI 77 recognizes a cryptic domain ofhuman collagen-I and IV that is also common to collagens II, III and V.This cryptic domain(s) is less accessible under most normalphysiological conditions but becomes accessible following proteolyticremodeling of the collagen triple helix in vivo. Thus, cryptic collagenepitope(s) can become more accessible during invasive cellularprocesses. Importantly, the cryptic domain(s) defined by theseantibodies was shown to be exposed within the basement membrane of tumorassociated angiogenic blood vessels from human tumors including, breast,bladder and melanoma tumors. However, this cryptic domain was lessexposed within the vessels or normal tissues tested. Therefore, theantibodies HUIV26 and HUI77 represent important and specific markers ofangiogenic blood vessels. These cryptic domain(s) plays an importantrole in regulating angiogenesis and tumor growth since the monoclonalantibodies HUIV26 and HUI77 potently inhibit angiogensis and human tumorgrowth in the chick embryo, rat and mouse models following systemicadministration (Xu et al., supra, 2001). Thus, these monoclonalantibodies and the antibodies of the invention having specific bindingactivity for these cryptic collagen site(s) represent a highly potentand effective new therapeutic reagent for the treatment for diseasescharacterized by aberrant neovascularization.

[0041] A nucleic acid sequence of the invention can include a sequencethat is the same or substantially the same as a specifically recited SEQID NO. Similarly, an amino acid sequence of the invention can include asequence that is the same or substantially the same as a specificallyrecited SEQ ID NO. As used herein, the term “substantially” or“substantially the same” when used in reference to a nucleotide or aminoacid sequence is intended to mean that the nucleotide or amino acidsequence shows a considerable degree, amount or extent of sequenceidentity when compared to a reference sequence, for example, thesequence of a parent antibody. Such a considerable degree, amount orextent of sequence identity is further considered to be significant andmeaningful and therefore exhibit characteristics which are definitivelyrecognizable or known. Thus, a nucleotide sequence which issubstantially the same nucleotide sequence as a heavy or light chain ofan antibody of the invention, including fragments thereof, refers to asequence which exhibits characteristics that are definitively known orrecognizable as encoding or as being the amino acid sequence as theparent antibody sequence. Minor modifications thereof are included solong as they are recognizable as a parent antibody sequence. Similarly,an amino acid sequence which is substantially the same amino acidsequence as a heavy or light chain of an antibody of the invention, orfunctional fragment thereof, refers to a sequence which exhibitscharacteristics that are definitively known or recognizable asrepresenting the amino acid sequence of parent antibody and minormodifications thereof. When determining whether a nucleotide or aminoacid sequence is substantially the same as a parent antibody,consideration is given to the number of changes relative to the parentantibody together with whether the function is maintained, for example,whether the function of binding to a cryptic collagen site is maintainedfor antibodies of the invention.

[0042] Minor modification of these nucleotide sequences and/or aminoacids are intended to be included as heavy and light chain encodingnucleic acids and their functional fragments. Such minor modificationsinclude, for example, those which do not change the encoded amino acidsequence due to the degeneracy of the genetic code as well as thosewhich result in only a conservative substitution of the encoded aminoacid sequence. Conservative substitutions of encoded amino acidsinclude, for example, amino acids which belong within the followinggroups: (1) non-polar amino acids (Gly, Ala, Val, Leu, and Ile); (2)polar neutral amino acids (Cys, Met, Ser, Thr, Asn, and Gln); (3) polaracidic amino acids (Asp and Glu); (4) polar basic amino acids (Lys, Argand His); and (5) aromatic amino acids (Phe, Trp, Tyr, and His). Otherminor modifications are included within the nucleic acids encoding heavyand light chain polypeptides of the invention so long as the nucleicacid or encoded polypeptides retain some or all of their function asdescribed herein.

[0043] To generate antibodies of the invention having specific bindingactivity for a cryptic collagen epitope, the heavy and light chainvariable regions of the antibodies HUIV26 and HUI77 were cloned andsequenced (see Example I and FIGS. 2 and 3). CDRs of the heavy and lightchain variable regions were identified. Exemplary heavy and light chainCDRs, as determined by the numbering of Kabat, are shown in FIGS. 2C and3C (underlined). Exemplary heavy and light chain CDRs of HUIV26 include,for example, V_(L) CDR1, KSSQSLLNSGNQKNYLA (SEQ ID NO:20); V_(L) CDR2,GASTRES (SEQ ID NO:22); V_(L) CDR3, QNDHSYPYT (SEQ ID NO:24); V_(H)CDR1, GFDFSRYWMS (SEQ ID NO:26); V_(H) CDR2, EINPDSSTINYTPSLKD (SEQ IDNO:28); and V_(H) CDR3, PVDGYYDAMDY (SEQ ID NO:30). Exemplary heavy andlight chain CDRs of HUI77 include, for example, V_(L) CDR1,RSSQSIVHSNGNTYLE (SEQ ID NO:32); V_(L) CDR2, KVSNRFS (SEQ ID NO:34);V_(L) CDR3, FQGSHVPWT (SEQ ID NO:36); V_(H) CDR1, GFSLSTSGMGVG (SEQ IDNO:38); V_(H) CDR2, DIWWDDNKYYNPSLKS (SEQ ID NO:40); and VH CDR3,RANYGNPYYAMDY (SEQ ID NO:42).

[0044] Libraries of CDR variants containing single amino acidsubstitutions were generated (Example II). The libraries were screenedfor binding to a cryptic collagen site, and single amino acid mutationshaving beneficial activity were identified. Combinatorial mutants, inwhich two or more variant CDRs containing at least one amino acidsubstitution relative to parental HUIV26 or HUI77 CDRs were combined andscreened for activity (Example III). A number of combinatorial mutantshaving optimized activity for binding to a cryptic collagen site wereidentified.

[0045] The antibodies of the invention having binding activity for acryptic collagen epitope. As disclosed herein, the collagen can bedenatured by any of a variety of methods so long as an antigenicdeterminant is exposed that was less accessible in native collagen. Suchmethods include, for example, proteolytic digestion, heat or thermaldenaturation, chemical denaturation, and the like. One skilled in theart will know a variety of methods suitable for denaturing a collagenmolecule to reveal a cryptic collagen site or epitope. Furthermore, themethod of denaturation can be a combination of two or more denaturationmethods, for example, proteolytic digestion combined with chemicaland/or thermal denaturation. For example, proteolytic digestion can beused to cleave collagen, resulting in a collagen molecule that is moresusceptible to thermal or chemical denaturation. An exemplary proteasethat can be used to denature collagen is matrix metalloproteinase, whichcan be used in vitro and can function in vivo to cleave collagen withintriple helical regions and at body temperature in a mammal.

[0046] The invention provides grafted antibodies of the HUIV26 and HUI77antibodies. In one embodiment, the invention provides a grafted antibodyof HUIV26. The grafted antibody, or functional fragment thereof,comprises one or more complementarity determining regions (CDRs) havingat least one amino acid substitution in one or more CDRs of a heavychain CDR selected from the group consisting of SEQ ID NOS:26, 28 and 30or a light chain CDR selected from the group consisting of SEQ IDNOS:20, 22 and 24, the grafted antibody or functional fragment thereofhaving specific binding activity for a cryptic collagen epitope.

[0047] In another embodiment, the invention provides a grafted antibodyof HUI77. The grafted antibody, or functional fragment thereof,comprises one or more complementarity determining regions (CDRs) havingat least one amino acid substitution in one or more CDRs of a heavychain CDR selected from the group consisting of SEQ ID NOS:38, 40 and 42or a light chain CDR selected from the group consisting of SEQ IDNOS:32, 34 and 36, the grafted antibody or functional fragment thereofhaving specific binding activity for a cryptic collagen epitope.

[0048] The invention additionally provides antibodies, or functionalfragments thereof, containing specifically recited CDRs, where theantibody or functional fragment thereof has specific binding activityfor a cryptic collagen epitope. Such antibodies include those having atleast a single amino acid substitution and which retain binding activityfor a cryptic collagen epitope. Included among such CDR variants arethose described in FIGS. 4 and 5.

[0049] Exemplary CDRs of the invention having a single amino acidsubstitution in a CDR of HUIV26 include, for example, those describedbelow, in which the position of the amino acid mutation in the numberingof Kabat is indicated along with the amino acid substitution from wildtype to mutant (wild type-mutant). Such exemplary CDRs include HuIV26V_(H) CDR1 31R→H (SEQ ID NO:43); HuIV26 V_(H) CDR1 34M→I (SEQ ID NO:44);HuIV26 V_(H) CDR1 35S→T (SEQ ID NO:45); HuIV26 V_(H) CDR1 35S→A (SEQ IDNO:46); HuIV26 V_(H) CDR1 35S→G (SEQ ID NO:47); HuIV26 V_(H) CDR2 57I→A(SEQ ID NO:48); HuIV26 V_(H) CDR2 57I→S (SEQ ID NO:49); HuIV26 V_(H)CDR2 62S→Y (SEQ ID NO:50); HuIV26 V_(H) CDR2 62S→A (SEQ ID NO:51);HuIV26 V_(H) CDR2 62S→H (SEQ ID NO:52); HuIV26 V_(H) CDR2 62S→G (SEQ IDNO:53); HuIV26 V_(H) CDR2 64K→Q (SEQ ID NO:54); HuIV26 V_(H) CDR2 65D→S(SEQ ID NO:55); HuIV26 V_(H) CDR3 97D→P (SEQ ID NO:56); HuIV26 V_(H)CDR3 97D→G (SEQ ID NO:57); HuIV26 V_(H) CDR3 97D→T (SEQ ID NO:58);HuIV26 V_(H) CDR3 97D→A (SEQ ID NO:59); HuIV26 V_(H) CDR3 98G→P (SEQ IDNO:60); HuIV26 V_(H) CDR3 98G→A (SEQ ID NO:61); HuIV26 V_(H) CDR3 98G→H(SEQ ID NO:62); HuIV26 V_(H) CDR3 102Y→P (SEQ ID NO:63); HuIV26 V_(H)CDR3 102Y→N (SEQ ID NO:64); HuIV26 V_(L) CDR1 27Q→R (SEQ ID NO:65);HuIV26 V_(L) CDR1 27Q→S (SEQ ID NO:66); HuIV26 V_(L) CDR1 27dN→S (SEQ IDNO:67); HuIV26 V_(L) CDR1 27eS→Y (SEQ ID NO:68); HuIV26 V_(L) CDR127eS→W (SEQ ID NO:69); HuIV26 V_(L) CDR1 27eS→H (SEQ ID NO:70); HuIV26V_(L) CDR1 27eS→R (SEQ ID NO:71); HuIV26 VL CDR1 27fG→Y (SEQ ID NO:72);HuIV26 V_(L) CDR1 27fG→R (SEQ ID NO:73); HuIV26 V_(L) CDR1 27fG→H (SEQID NO:74); HuIV26 V_(L) CDR1 27fG→I (SEQ ID NO:75); HuIV26 V_(L) CDR129Q→K (SEQ ID NO:76); HuIV26 V_(L) CDR3 93S→Q (SEQ ID NO:77); HuIV26 VLCDR3 93S→G (SEQ ID NO:78); HuIV26 V_(L) CDR3 93S→L (SEQ ID NO:79);HuIV26 V_(L) CDR3 93S→A (SEQ ID NO:80); HuIV26 V_(L) CDR3 93S→T (SEQ IDNO:81); HuIV26 V_(L) CDR3 93S→V (SEQ ID NO:82); HuIV26 V_(L) CDR3 94Y→N(SEQ ID NO:83); HuIV26 V_(L) CDR3 94Y→S (SEQ ID NO:84); HuIV26 V_(L)CDR3 94Y→P (SEQ ID NO:85); HuIV26 V_(L) CDR3 94Y→M (SEQ ID NO:86); andHuIV26 V_(L) CDR2 57I→V (SEQ ID NO:162).

[0050] Exemplary CDRs of the invention having a single amino acidsubstitution in a CDR of HUI77 include, for example, those describedbelow, in which the position of the amino acid mutation in the numberingof Kabat is indicated along with the amino acid substitution from wildtype to mutant (wild type-mutant). Such exemplary CDRs include HUI77V_(H) CDR1 32S→P (SEQ ID NO:87); HUI77 V_(H) CDR1 32S→W (SEQ ID NO:88);HUI77 V_(H) CDR1 35bG→W (SEQ ID NO:89); HUI77 V_(H) CDR1 35bG→L (SEQ IDNO:90); HUI77 V_(H) CDR1 35bG→A (SEQ ID NO:91); HUI77 V_(H) CDR2 59Y→S(SEQ ID NO:92); HUI77 V_(H) CDR2 59Y→A (SEQ ID NO:93); HUI77 V_(H) CDR259Y→P (SEQ ID NO:94); HUI77 V_(H) CDR2 64K→P (SEQ ID NO:95); HUI77 V_(H)CDR3 95R→P (SEQ ID NO:96); HUI77 V_(H) CDR3 95R→Q (SEQ ID NO:97); HUI77V_(H) CDR3 95R→L (SEQ ID NO:98); HUI77 V_(H) CDR3 95R→T (SEQ ID NO:99);HUI77 V_(H) CDR3 95R→V (SEQ ID NO:100); HUI77 V_(H) CDR3 100N→V (SEQ IDNO:101); HUI77 V_(H) CDR3 100N→W (SEQ ID NO:102); HUI77 V_(H) CDR3100eM→Q (SEQ ID NO:103); HUI77 V_(H) CDR3 100eM→N (SEQ ID NO:104); HUI77V_(H) CDR3 100eM→T (SEQ ID NO:105); HUI77 V_(H) CDR3 102Y→K (SEQ IDNO:106); HUI77 V_(L) CDR3 102Y→T (SEQ ID NO:107); HUI77 V_(H) CDR3102Y→M (SEQ ID NO:108); HUI77 V_(H) CDR3 102Y→H (SEQ ID NO:109); HUI77V_(L) CDR1 27cV→P (SEQ ID NO:110); HUI77 V_(L) CDR1 27cV→W (SEQ IDNO:111); HUI77 V_(L) CDR1 27dH→L (SEQ ID NO:112); HUI77 V_(L) CDR127dH→S (SEQ ID NO:113); HUI77 V_(L) CDR1 27eS→W (SEQ ID NO:114); HUI77V_(L) CDR1 28N→Y (SEQ ID NO:115); HUI77 V_(L) CDR1 28N→W (SEQ IDNO:116); HUI77 V_(L) CDR1 30N→Y (SEQ ID NO:117); HUI77 V_(L) CDR1 33L→F(SEQ ID NO:118); HUI77 V_(L) CDR1 33L→V (SEQ ID NO:119); HUI77 V_(L)CDR2 50K→S (SEQ ID NO:120); HUI77 V_(L) CDR2 51V→A (SEQ ID NO:121);HUI77 V_(L) CDR2 53N→S (SEQ ID NO:122); HUI77 V_(L) CDR2 54R→L (SEQ IDNO:123); HUI77 V_(L) CDR2 56S→W (SEQ ID NO:124); HUI77 V_(L) CDR2 56S→F(SEQ ID NO:125); HUI77 V_(L) CDR3 89F→V (SEQ ID NO:126); HUI77 V_(L)CDR3 89F→H (SEQ ID NO:127); HUI77 V_(L) CDR3 90Q→R (SEQ ID NO:128);HUI77 V_(L) CDR3 90Q→W (SEQ ID NO:129); HUI77 VL CDR3 91G→S (SEQ IDNO:130); HUI77 V_(L) CDR3 92S→W (SEQ ID NO:131); HUI77 V_(L) CDR3 92S→E(SEQ ID NO:132); HUI77 V_(L) CDR3 93H→L (SEQ ID NO:133); HUI77 V_(L)CDR3 93H→T (SEQ ID NO:134); HUI77 V_(L) CDR3 93H→S (SEQ ID NO:135);HUI77 V_(L) CDR3 93H→A (SEQ ID NO:136); HUI77 V_(L) CDR3 93H→Q (SEQ IDNO:137); HUI77 V_(L) CDR3 94V→T (SEQ ID NO:138); HUI77 V_(L) CDR3 97T→A(SEQ ID NO:139); HUI77 V_(L) CDR3 97T→R (SEQ ID NO:140); HUI77 V_(L)CDR3 97T→H (SEQ ID NO:141); HUI77 V_(L) CDR3 97T→K (SEQ ID NO:142);HUI77 V_(L) CDR3 97T→I (SEQ ID NO:143); HUI77 V_(H) CDR2 59Y→T (SEQ IDNO:144); HUI77 V_(L) CDR3 94V→F (SEQ ID NO:145); and HUI77 V_(L) CDR128N→Q (SEQ ID NO:146)

[0051] In addition to CDRs having single amino acid substitutions, theinvention additionally provides HUIV26 and HUI77 CDRs having two or moreamino acid substitutions. Exemplary CDRs having two or more amino acidsubstitutions in HUIV26 include, for example, HUIV26 V_(H) CDR257I→A/62S→A (SEQ ID NO:154); HUIV26 V_(H) CDR2 57I→A/62S→Y (SEQ IDNO:155); HUIV26 V_(H) CDR2 57I→A/62S→H (SEQ ID NO:156); HUIV26 V_(L)CDR1 27eS→W/27fG→Y (SEQ ID NO:157); HUIV26 V_(L) CDR1 27eS→Y/27fG→Y (SEQID NO:158); HUIV26 V_(L) CDR1 27eS→Y/27fG→H (SEQ ID NO:159); HUIV26V_(L) CDR1 27eS→R/27fG→Y (SEQ ID NO:160); and HUIV26 V_(L) CDR127eS→W/27fG→H (SEQ ID NO:161) (see FIG. 6). Exemplary CDRs having two ormore amino acid substitutions in HUI77 include, for example, HUI77 V_(H)CDR1 32S→P/35bG→W (SEQ ID NO:147); HUI77 V_(H) CDR1 32S→P/35bG→A (SEQ IDNO:148); HUI77 V_(L) CDR1 27dH→S/28N→W (SEQ ID NO:149); HUI77 V_(L) CDR127dH→S/28N→Y (SEQ ID NO:150); HUI77 V_(L) CDR1 27d→HS/28N→Q (SEQ IDNO:151); HUI77 V_(L) CDR1 28N→Q/33L→F (SEQ ID NO:152); HUI77 V_(L) CDR127H→S/28N→W/33L→F (SEQ ID NO:153); and HUI77 V_(L) CDR3 91G→S/94V→F (SEQID NO:358) (see FIG. 7).

[0052] The invention provides an antibody having at least one of theabove variant CDR sequences. It is understood that any combination ofHUIV26 CDRs can be combined with mutant and/or wild type CDRs togenerate an HUIV26 grafted antibody, so long as binding activity to acryptic collagen site is maintained. Similarly, any combination of HUI77CDRs can be combined with mutant and/or wild type CDRs to generate aHUI77 grafted antibody so long as binding activity to a cryptic collagensite is maintained. Thus, any combination of single amino acidsubstitutions can be combined with other CDR mutants to generate anantibody having at least two variant CDRs. Furthermore, any singlemutation at different positions within the same CDR can be combined togenerate a CDR having 2 or more amino acid substitutions at two or morepositions. Any of the single or multiple mutations can be combined solong as binding activity to a cryptic collagen site is maintained.

[0053] Thus, the invention provides an antibody, or functional fragmentthereof, comprising one or more CDRs selected from the group consistingof CDRs referenced as SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:78, SEQ ID NO:77, SEQ ID NO:78; SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ IDNO:86, SEQ ID NO: 154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, and SEQ IDNO:162, the antibody or functional fragment thereof having specificbinding activity for a cryptic collagen epitope.

[0054] The invention additionally provides an antibody, or functionalfragment thereof, comprising one or more CDRs selected from the groupconsisting of CDRs referenced as SEQ ID NO:87, SEQ ID NO:88, SEQ IDNO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ IDNO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ IDNO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108,SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ IDNO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122,SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ IDNO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136,SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ IDNO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150,SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, and SED ID NO:358 theantibody or functional fragment thereof having specific binding activityfor a cryptic collagen epitope.

[0055] The invention further provides an antibody, or functionalfragment thereof, comprising a heavy chain polypeptide comprising one ormore CDRs having at least one amino acid substitution in one or moreheavy chain CDRs, the heavy chain CDRs selected from the groupconsisting of a heavy chain CDR1 selected from the group consisting ofCDRs referenced as SEQ ID NOS:26, 43, 44, 45, 46, and 47; a heavy chainCDR2 selected from the group consisting of CDRs referenced as SEQ IDNOS:28, 48, 49, 50, 51, 52, 53, 54, and 55; and a heavy chain CDR3selected from the group consisting of CDRs referenced as SEQ ID NOS:30,56, 57, 58, 59, 60, 61, 62, 63, and 64, the antibody or functionalfragment thereof having specific binding activity for a cryptic collagenepitope.

[0056] The invention also provides an antibody, or functional fragmentthereof, comprising a light chain polypeptide comprising one or moreCDRs having at least one amino acid substitution in one or more lightchain CDRs, the light chain CDRs selected from the group consisting of alight chain CDR1 selected from the group consisting of CDRs referencedas SEQ ID NOS:20, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, and 76; alight chain CDR2 referenced as SEQ ID NO:22:; and a light chain CDR3selected from the group consisting of CDRs referenced as SEQ ID NOS:24,77, 78, 79, 80, 81, 82, 83, 84, 85, and 86, the antibody or functionalfragment thereof having specific binding activity for a cryptic collagenepitope.

[0057] The invention further provides an antibody, or functionalfragment thereof, comprising a heavy chain polypeptide comprising one ormore CDRs having at least one amino acid substitution in one or moreheavy chain CDRs, the heavy chain CDRs selected from the groupconsisting of a heavy chain CDR1 selected from the group consisting ofCDRs referenced as SEQ ID NOS:38, 87, 88, 89, 90, 91, 147 and 148; aheavy chain CDR2 selected from the group consisting of CDRs referencedas SEQ ID NOS:40, 92, 93, 94, 95 and 144; and a heavy chain CDR3selected from the group consisting of CDRs referenced as SEQ ID NOS:42,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108 and 109, theantibody or functional fragment thereof having specific binding activityfor a cryptic collagen epitope.

[0058] Additionally provided is an antibody, or functional fragmentthereof, comprising a light chain polypeptide comprising one or moreCDRs having at least one amino acid substitution in one or more lightchain CDRs, the light chain CDRs selected from the group consisting of alight chain CDR1 selected from the group consisting of CDRs referencedas SEQ ID NOS:32, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 146,149, 150, 151, 152 and 153; a light chain CDR2 referenced as SEQ IDNOS:34, 120, 121, 122, 123, 124 and 125; and a light chain CDR3 selectedfrom the group consisting of CDRs referenced as SEQ ID NOS:36, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 145 and 358, the antibody or functional fragment thereofhaving specific binding activity for a cryptic collagen epitope.

[0059] As described above, an antibody of the invention can be generatedfrom any combination of the variant and/or wild type CDRs, so long asbinding activity to a cryptic collagen site is maintained. As disclosedherein, a variety of combinatorial antibodies containing multiple CDRshaving at least a single amino acid substitution were identified havingbinding activity for a cryptic collagen site. In addition to antibodiescontaining any combination of the respective CDRs disclosed herein, thefollowing specific combinations of CDRs are also provided by theinvention.

[0060] Exemplary HUIV26 variants include, for example, the followingantibodies:

[0061] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:26; a heavy chain CDR2 referenced as SEQ ID NO:28; a heavy chain CDR3referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:20; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (4.1-2D4).

[0062] An antibody comprises a heavy chain CDR1 referenced as SEQ IDNO:26; a heavy chain CDR2 referenced as SEQ ID NO:28; a heavy chain CDR3referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:72; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (L1b-F11).

[0063] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:26; a heavy chain CDR2 referenced as SEQ ID NO:48; a heavy chain CDR3referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:20; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (H2a-G8).

[0064] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:45; a heavy chain CDR2 referenced as SEQ ID NO:154; a heavy chainCDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:157; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (DcomA2).

[0065] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:26; a heavy chain CDR2 referenced as SEQ ID NO:155; a heavy chainCDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:158; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (DcomA4).

[0066] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:46; a heavy chain CDR2 referenced as SEQ ID NO:155; a heavy chainCDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:159; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (DcomB1).

[0067] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:26; a heavy chain CDR2 referenced as SEQ ID NO:48; a heavy chain CDR3referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:160; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (DcomD2).

[0068] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:45; a heavy chain CDR2 referenced as SEQ ID NO:155; a heavy chainCDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:72; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (DcomD3).

[0069] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:26; a heavy chain CDR2 referenced as SEQ ID NO:155; a heavy chainCDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:157; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (DcomD6).

[0070] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:45; a heavy chain CDR2 referenced as SEQ ID NO:155; a heavy chainCDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:160; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (DcomE3).

[0071] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:46; a heavy chain CDR2 referenced as SEQ ID NO:155; a heavy chainCDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:160; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (DcomG2).

[0072] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:45; a heavy chain CDR2 referenced as SEQ ID NO:162; a heavy chainCDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:158; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (DcomA7).

[0073] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:45; a heavy chain CDR2 referenced as SEQ ID NO:156; a heavy chainCDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:157; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (DcomB10).

[0074] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:26; a heavy chain CDR2 referenced as SEQ ID NO:154; a heavy chainCDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:157; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (DcomC8).

[0075] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:45; a heavy chain CDR2 referenced as SEQ ID NO:155; a heavy chainCDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:157; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (DcomD7).

[0076] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:46; a heavy chain CDR2 referenced as SEQ ID NO:154; a heavy chainCDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:161; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (DcomD11).

[0077] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:46; a heavy chain CDR2 referenced as SEQ ID NO:156; a heavy chainCDR3 referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:161; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (DcomE11).

[0078] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:46; a heavy chain CDR2 referenced as SEQ ID NO:28; a heavy chain CDR3referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:20; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77 (2D4H1-C3).

[0079] Exemplary HUI77 variants include, for example, the followingantibodies:

[0080] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:38; a heavy chain CDR2 referenced as SEQ ID NO:40; a heavy chain CDR3referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQ IDNO:32; a light chain CDR2 referenced as SEQ ID NO:34; and a light chainCDR3 referenced as SEQ ID NO:36 (12F10Q).

[0081] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:38; a heavy chain CDR2 referenced as SEQ ID NO:92; a heavy chain CDR3referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQ IDNO:32; a light chain CDR2 referenced as SEQ ID NO:34; and a light chainCDR3 referenced as SEQ ID NO:36 (QH2b-A3).

[0082] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:147; a heavy chain CDR2 referenced as SEQ ID NO:92; a heavy chainCDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQID NO:149; a light chain CDR2 referenced as SEQ ID NO:34; and a lightchain CDR3 referenced as SEQ ID NO:36 (Qcom1B6).

[0083] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:147; a heavy chain CDR2 referenced as SEQ ID NO:92; a heavy chainCDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQID NO:150; a light chain CDR2 referenced as SEQ ID NO:34; and a lightchain CDR3 referenced as SEQ ID NO:36 (Qcom1B8).

[0084] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:147; a heavy chain CDR2 referenced as SEQ ID NO:93; a heavy chainCDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQID NO:149; a light chain CDR2 referenced as SEQ ID NO:34; and a lightchain CDR3 referenced as SEQ ID NO:36 (Qcom1C3).

[0085] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:147; a heavy chain CDR2 referenced as SEQ ID NO:144; a heavy chainCDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQID NO:149; a light chain CDR2 referenced as SEQ ID NO:34; and a lightchain CDR3 referenced as SEQ ID NO:36 (Qcom1D3).

[0086] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:147; a heavy chain CDR2 referenced as SEQ ID NO:93; a heavy chainCDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQID NO:151; a light chain CDR2 referenced as SEQ ID NO:34; and a lightchain CDR3 referenced as SEQ ID NO:36 (Qcom1E3).

[0087] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:147; a heavy chain CDR2 referenced as SEQ ID NO:92; a heavy chainCDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQID NO:151; a light chain CDR2 referenced as SEQ ID NO:34; and a lightchain CDR3 referenced as SEQ ID NO:36 (Qcom1H6).

[0088] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:147; a heavy chain CDR2 referenced as SEQ ID NO:93; a heavy chainCDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQID NO:152; a light chain CDR2 referenced as SEQ ID NO:34; and a lightchain CDR3 referenced as SEQ ID NO:145 (Qcom1H7).

[0089] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:148; a heavy chain CDR2 referenced as SEQ ID NO:93; a heavy chainCDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQID NO:150; a light chain CDR2 referenced as SEQ ID NO:34; and a lightchain CDR3 referenced as SEQ ID NO:36 (Qcom2A4).

[0090] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:147; a heavy chain CDR2 referenced as SEQ ID NO:93; a heavy chainCDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQID NO:115; a light chain CDR2 referenced as SEQ ID NO:34; and a lightchain CDR3 referenced as SEQ ID NO:36 (Qcom2B11).

[0091] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:147; a heavy chain CDR2 referenced as SEQ ID NO:40; a heavy chainCDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQID NO:153; a light chain CDR2 referenced as SEQ ID NO:34; and a lightchain CDR3 referenced as SEQ ID NO:36 (Qcom2C1).

[0092] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:147; a heavy chain CDR2 referenced as SEQ ID NO:92; a heavy chainCDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQID NO:116; a light chain CDR2 referenced as SEQ ID NO:34; and a lightchain CDR3 referenced as SEQ ID NO:36 (Qcom2D9).

[0093] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:147; a heavy chain CDR2 referenced as SEQ ID NO:93; a heavy chainCDR3 referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQID NO:116; a light chain CDR2 referenced as SEQ ID NO:34; and a lightchain CDR3 referenced as SEQ ID NO:36 (Qcom2E3).

[0094] An antibody comprising a heavy chain CDR1 referenced as SEQ IDNO:38; a heavy chain CDR2 referenced as SEQ ID NO:93; a heavy chain CDR3referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQ IDNO:32; a light chain CDR2 referenced as SEQ ID NO:34; and a light chainCDR3 referenced as SEQ ID NO:130 (Qh2b-B7).

[0095] The invention also provides grafted antibodies containing CDRsderived from HUIV26 and HUI77, respectively. Such grafted CDRs includehumanized antibodies, in which CDRs from HUIV26 or HUI77 have beengrafted or in which a CDR containing one or more amino acidsubstitutions is grafted. The CDRs can be grafted directly into a humanframework, as disclosed herein. If desired, framework changes can alsobe incorporated by generating framework libraries. The optimization ofCDRs and/or framework sequences can be performed independently andsequentially combined or can be performed simultaneously, as describedin more detail below.

[0096] Thus, the invention additionally provides a grafted antibody inwhich HUIV26 CDRs (SEQ ID NOS:20, 22, 24, 26, 28 and 30) are graftedinto a human framework sequence. Also provided is a grafted antibody inwhich HUI77 CDRs (SEQ ID NOS:32, 34, 36, 38, 40 and 42) are grafted intoa human framework.

[0097] To generate grafted antibodies, donor CDRs of collagen-specificantibodies are grafted onto an antibody acceptor variable regionframework. Methods for grafting antibodies and generating CDR variantsto optimize activity have been described previously (WO 98/33919; WO00/78815; WO 01/27160). The procedure can be performed to achievegrafting of donor CDRs and affinity reacquisition in a simultaneousprocess. The methods similarly can be used, either alone or incombination with CDR grafting, to modify or optimize the bindingaffinity of a variable region. The methods for conferring donor CDRbinding affinity onto an acceptor variable region are applicable to bothheavy and light chain variable regions and as such can be used tosimultaneously graft and optimize the binding affinity of an antibodyvariable region.

[0098] The donor CDRs can be altered to contain a plurality of differentamino acid residue changes at all or selected positions within the donorCDRs. For example, random or biased incorporation of the twentynaturally occurring amino acid residues, or preselected subsets, can beintroduced into the donor CDRs to produce a diverse population of CDRspecies. Inclusion of CDR variant species into the diverse population ofvariable regions allows for the generation of variant species thatexhibit optimized binding affinity for a predetermined antigen.

[0099] A range of possible changes can be made in the donor CDRpositions. Some or all of the possible changes that can be selected forchange can be introduced into the population of grafted donor CDRs. Asingle position in a CDR can be selected to introduce changes or avariety of positions having altered amino acids can be combined andscreened for activity.

[0100] One approach is to change all amino acid positions along a CDR byreplacement at each position with, for example, all twenty naturallyoccurring amino acids. The replacement of each position can occur in thecontext of other donor CDR amino acid positions so that a significantportion of the CDR maintains the authentic donor CDR sequence, andtherefore, the binding affinity of the donor CDR. For example, anacceptor variable region framework, either a native or alteredframework, can be grafted with a population of CDRs containing singleposition replacements at each position within the CDRs. Similarly, anacceptor variable region framework can be targeted for grafting with apopulation of CDRs containing more than one position changed toincorporate all twenty amino acid residues, or a subset of amino acids.One or more amino acid positions within a CDR, or within a group of CDRsto be grafted, can be altered and grafted into an acceptor variableregion framework to generate a population of grafted antibodies. It isunderstood that a CDR having one or more altered positions can becombined with one or more other CDRs having one or more alteredpositions, if desired.

[0101] A population of CDR variant species having one or more alteredpositions can be combined with any or all of the CDRs which constitutethe binding pocket of a variable region. Therefore, an acceptor variableregion framework can be targeted for the simultaneous incorporation ofdonor CDR variant populations at one, two or all three recipient CDRlocations in a heavy or light chain. The choice of which CDR or thenumber of CDRs to target with amino acid position changes will dependon, for example, if a full CDR grafting into an acceptor is desired orwhether the method is being performed for optimization of bindingaffinity.

[0102] Another approach for selecting donor CDR amino acids to changefor conferring donor CDR binding affinity onto an antibody acceptorvariable region framework is to select known or readily identifiable CDRpositions that are highly variable. For example, the variable regionCDR3 is generally highly variable. This region therefore can beselectively targeted for amino acid position changes during graftingprocedures to ensure binding affinity reacquisition or augmentation,either alone or together with relevant acceptor variable frameworkchanges, as described herein.

[0103] If desired, CDR variant populations having one or more alteredamino acid positions can be advantageously combined with a frameworkvariant population having one or more altered amino acid positions. Sucha combination can result in beneficial combinations of changes, whichare identified by screening for an optimized activity.

[0104] The resultant population of CDR grafted variable regionstherefore contain a species corresponding to the authentic parent aminoacid residue at each position as well as a diverse number of differentspecies which correspond to the possible combinations and permutationsof the authentic parent amino acid residues together with the variantresidues at each of the selected CDR positions. Such a diversepopulation of CDR grafted variable regions are screened for an alteredvariable region species which retains donor CDR binding activity, orwhich has optimized binding activity.

[0105] An acceptor can be selected so that it is closely similar to thevariable region amino acid sequence harboring the donor CDRs. Inaddition, a variety of acceptors less closely related to the donorantibody can be used. Alternatively, a library of all possible orrelevant changes in the acceptor framework can be made and then screenedfor those variable regions, or heteromeric binding fragments thereof,that maintain or exhibit increased binding affinity compared to thedonor molecule. The donor CDRs can be grafted into a variety ofnaturally occurring acceptor frameworks or altered frameworks having oneor more changes or even a library containing changes at one or morepositions. Therefore, the applicability is not preconditioned on theavailability or search for an acceptor framework variable region similarto that of the donor.

[0106] The methods for conferring donor CDR binding affinity onto avariable region can involve identifying the relevant amino acidpositions in the acceptor framework that are known or predicted toinfluence a CDR conformation, or that are known or predicted toinfluence the spacial context of amino acid side chains within the CDRthat participate in binding, and then generating a population of alteredvariable region species that incorporate a plurality of different aminoacid residues at those positions. For example, the different amino acidresidues at those positions can be incorporated either randomly or witha predetermined bias and can include all of the twenty naturallyoccurring amino acid residues at each of the relevant positions.Subsets, including less than all of the naturally occurring amino acidscan additionally be chosen for incorporation at the relevant frameworkpositions. Including a plurality of different amino acid residues ateach of the relevant framework positions ensures that there will be atleast one species within the population that will have framework changeswhich allows the CDRs to reacquire their donor binding affinity in thecontext of the acceptor framework variable region.

[0107] For humanizing an antibody, any of a variety of human frameworkscan be selected for CDR grafting. For example, CDRs of HUIV26 or HUI77can be cloned into a variety of human framework sequences. Theframeworks can be generated using human germline genes encoding heavyand light chain variable regions as well as J regions to obtain humanframework sequences for CDR grafting. Exemplary human frameworknucleotide sequences include, for example, the framework sequences ofDPK24 (VKIV) (SEQ ID NO:5), DP-54 (VHIII) (SEQ ID NO:7), DPK13 (VKII)(SEQ ID NO:13), DP-28 (VHII) (SEQ ID NO:15), as well as J regions JK1(SEQ ID NO:217), JK2 (SEQ ID NO:218) and JH6 (SEQ ID NO:219). It isunderstood that framework regions from any available germline sequencecan be combined with any available J sequence, as desired, to generate ahuman framework for grafting CDRs. For example, an alignment of mousevariable regions of HUIV26 and HUI77 with an exemplary human frameworkis shown in FIGS. 2C and 3C, respectively. A fusion of VKIV/JK2 lightchain variable region and VHIII/JH6 heavy chain variable region arealigned with HUIV26 (FIG. 2C). A fusion of VKII/JK1 light chain variableregion and VHIII/JH6 heavy chain variable region are aligned with HUI77(FIG. 3C). An exemplary fusion of a germline and J region is shown inFIG. 3D, which is aligned with the HUI77 light chain. It is understoodthat any available human framework can be selected for CDR grafting and,if desired, optimized by the methods disclosed herein. As disclosedherein, CDRs having beneficial mutations can be grafted into a varietyof frameworks and have retained or improved activity (see Example III).

[0108] Selection of the relevant framework amino acid positions to alterdepends on a variety of criteria well known to those skilled it the art.One criteria for selecting relevant framework amino acids to change canbe the relative differences in amino acid framework residues between thedonor and acceptor molecules. Selection of relevant framework positionsto alter using this approach is simple and has the advantage of avoidingany subjective bias in residue determination or any bias in CDR bindingaffinity contribution by the residue.

[0109] Another criteria that can be used for determining the relevantamino acid positions to change can be, for example, selection offramework residues that are known to be important or to contribute toCDR conformation. For example, canonical framework residues areimportant for CDR conformation or structure. Targeting of a canonicalframework residue as a relevant position to change can identify a morecompatible amino acid residue in context with its associated donor CDRsequence.

[0110] The frequency of an amino acid residue at a particular frameworkposition is another criteria which can be used for selecting relevantframework amino acid positions to change. For example, comparison of theselected framework with other framework sequences within its subfamilycan reveal residues that occur at minor frequences at a particularposition or positions. Such positions harboring less abundant residuesare similarly applicable for selection as a position to alter in theacceptor variable region framework.

[0111] The relevant amino acid positions to change also can be selected,for example, based on proximity to a CDR. In certain contexts, suchresidues can participate in CDR conformation or antigen binding.Moreover, this criteria can similarly be used to prioritize relevantpositions selected by other criteria described herein. Therefore,differentiating between residues proximal and distal to one or more CDRsis an efficient way to reduce the number of relevant positions tochange.

[0112] Other criteria for selecting relevant amino acid frameworkpositions to alter include, for example, residues that are known orpredicted to reside in three-dimensional space near the antigen-CDRinterface or predicted to modulate CDR activity. Similarly, frameworkresidues that are known or predicted to form contacts between the heavy(V_(H)) and light (V_(L)) chain variable region interface can beselected. Such framework positions can affect the conformation oraffinity of a CDR by modulating the CDR binding pocket, antigeninteraction or the V_(H) and V_(L) interaction. Therefore, selection ofthese amino acid positions for constructing a diverse population forscreening of binding activity can be used to identify framework changeswhich replace residues having detrimental effects on CDR conformation orcompensate for detrimental effects of residues occurring elsewhere inthe framework.

[0113] Other framework residues that can be selected for alterationinclude amino acid positions that are inaccessible to solvent. Suchresidues are generally buried in the variable region and are thereforecapable of influencing the conformation of the CDR or V_(H) and V_(L)interactions. Solvent accessibility can be predicted, for example, fromthe relative hydrophobicity of the environment created by the amino acidside chains of the polypeptide or by known three-dimensional structuraldata.

[0114] Following selection of relevant amino acid positions in the donorCDRs, as well as any relevant amino acid positions in the frameworkregions desired to be varied, amino acid changes at some or all of theselected positions can be incorporated into encoding nucleic acids forthe acceptor variable region framework and donor CDRs. Altered frameworkor CDR sequences can be individually made and tested, or can besimultaneously combined and tested, if desired.

[0115] The variability at any or all of the altered positions can rangefrom a few to a plurality of different amino acid residues, includingall twenty naturally occurring amino acids or functional equivalents andanalogues thereof.

[0116] Selection of the number and location of the amino acid positionsto vary is flexible and can depend on the intended use and desiredefficiency for identification of the altered variable region having adesirable activity such as substantially the same or greater bindingaffinity compared to the donor variable region. In this regard, thegreater the number of changes that are incorporated into a alteredvariable region population, the more efficient it is to identify atleast one species that exhibits a desirable activity, for example,substantially the same or greater binding affinity as the donor.Alternatively, where the user has empirical or actual data to the affectthat certain amino acid residues or positions contributedisproportionally to binding affinity, then it can be desirable toproduce a limited population of altered variable regions which focuseson changes within or around those identified residues or positions.

[0117] For example, if CDR grafted variable regions are desired, alarge, diverse population of altered variable regions can include allthe non-identical framework region positions between the donor andacceptor framework and all single CDR amino acid position changes.Alternatively, a population of intermediate diversity can includesubsets, for example, of only the proximal non-identical frameworkpositions to be incorporated together with all single CDR amino acidposition changes. The diversity of the above populations can be furtherincreased by, for example, additionally including all pairwise CDR aminoacid position changes. In contrast, populations focusing onpredetermined residues or positions which incorporate variant residuesat as few as one framework and/or one CDR amino acid position cansimilarly be constructed for screening and identification of an alteredantibody variable region of the invention. As with the abovepopulations, the diversity of such focused populations can be furtherincreased by additionally expanding the positions selected for change toinclude other relevant positions in either or both of the framework andCDR regions. There are numerous other combinations ranging from fewchanges to many changes in either or both of the framework regions andCDRs that can additionally be employed, all of which will result in apopulation of altered variable regions that can be screened for theidentification of at least one CDR grafted altered variable regionhaving desired activity, for example, binding activity to a crypticcollagen site. Those skilled in the art will know, or can determine,which selected residue positions in the framework or donor CDRs, orsubsets thereof, can be varied to produce a population for screening andidentification of an altered antibody of the invention given theteachings and guidance provided herein.

[0118] Simultaneous incorporation of all of the CDR encoding nucleicacids and all of the selected amino acid position changes can beaccomplished by a variety of methods known to those skilled in the art,including for example, recombinant and chemical synthesis. For example,simultaneous incorporation can be accomplished by, for example,chemically synthesizing the nucleotide sequence for the acceptorvariable region, fused together with the donor CDR encoding nucleicacids, and incorporating at the positions selected for harboringvariable amino acid residues a plurality of corresponding amino acidcodons.

[0119] One such method well known in the art for rapidly and efficientlyproducing a large number of alterations in a known amino acid sequenceor for generating a diverse population of variable or random sequencesis known as codon-based synthesis or mutagenesis. This method is thesubject matter of U.S. Pat. Nos. 5,264,563 and 5,523,388 and is alsodescribed in Glaser et al. J. Immunology 149:3903 (1992). Briefly,coupling reactions for the randomization of, for example, all twentycodons which specify the amino acids of the genetic code are performedin separate reaction vessels and randomization for a particular codonposition occurs by mixing the products of each of the reaction vessels.Following mixing, the randomized reaction products corresponding tocodons encoding an equal mixture of all twenty amino acids are thendivided into separate reaction vessels for the synthesis of eachrandomized codon at the next position. For the synthesis of equalfrequencies of all twenty amino acids, up to two codons can besynthesized in each reaction vessel.

[0120] Variations to these synthesis methods also exist and include forexample, the synthesis of predetermined codons at desired positions andthe biased synthesis of a predetermined sequence at one or more codonpositions. Biased synthesis involves the use of two reaction vesselswhere the predetermined or parent codon is synthesized in one vessel andthe random codon sequence is synthesized in the second vessel. Thesecond vessel can be divided into multiple reaction vessels such as thatdescribed above for the synthesis of codons specifying totally randomamino acids at a particular position. Alternatively, a population ofdegenerate codons can be synthesized in the second reaction vessel suchas through the coupling of NNG/T nucleotides where N is a mixture of allfour nucleotides. Following synthesis of the predetermined and randomcodons, the reaction products in each of the two reaction vessels aremixed and then redivided into an additional two vessels for synthesis atthe next codon position.

[0121] A modification to the above-described codon-based synthesis forproducing a diverse number of variant sequences can similarly beemployed for the production of the variant populations described herein.This modification is based on the two vessel method described above,which biases synthesis toward the parent sequence and allows the user toseparate the variants into populations containing a specified number ofcodon positions that have random codon changes.

[0122] Briefly, this synthesis is performed by continuing to divide thereaction vessels after the synthesis of each codon position into two newvessels. After the division, the reaction products from each consecutivepair of reaction vessels, starting with the second vessel, is mixed.This mixing brings together the reaction products having the same numberof codon positions with random changes. Synthesis proceeds by thendividing the products of the first and last vessel and the newly mixedproducts from each consecutive pair of reaction vessels and redividinginto two new vessels. In one of the new vessels, the parent codon issynthesized and in the second vessel, the random codon is synthesized.For example, synthesis at the first codon position entails synthesis ofthe parent codon in one reaction vessel and synthesis of a random codonin the second reaction vessel. For synthesis at the second codonposition, each of the first two reaction vessels is divided into twovessels yielding two pairs of vessels. For each pair, a parent codon issynthesized in one of the vessels and a random codon is synthesized inthe second vessel. When arranged linearly, the reaction products in thesecond and third vessels are mixed to bring together those productshaving random codon sequences at single codon positions. This mixingalso reduces the product populations to three, which are the startingpopulations for the next round of synthesis. Similarly, for the third,fourth and each remaining position, each reaction product population forthe preceding position are divided and a parent and random codonsynthesized.

[0123] Following the above modification of codon-based synthesis,populations containing random codon changes at one, two, three and fourpositions as well as others can be conveniently separated out and usedbased on the need of the individual. Moreover, this synthesis schemealso allows enrichment of the populations for the randomized sequencesover the parent sequence since the vessel containing only the parentsequence synthesis is similarly separated out from the random codonsynthesis.

[0124] Other methods well known in the art for producing a large numberof alterations in a known amino acid sequence or for generating adiverse population of variable or random sequences include, for example,degenerate or partially degenerate oligonucleotide synthesis. Codonsspecifying equal mixtures of all four nucleotide monomers, representedas NNN, results in degenerate synthesis. Whereas partially degeneratesynthesis can be accomplished using, for example, the NNG/T codondescribed previously. Other methods well known in the art canalternatively be used such as the use of statistically predetermined, orvarigated, codon synthesis, which is the subject matter of U.S. Pat.Nos. 5,223,409 and 5,403,484.

[0125] Once the populations of altered variable region encoding nucleicacids have been constructed as described above, they can be expressed togenerate a population of altered variable region polypeptides that canbe screened for binding affinity. For example, the altered variableregion encoding nucleic acids can be cloned into an appropriate vectorfor propagation, manipulation and expression. Such vectors are known orcan be constructed by those skilled in the art and should contain allexpression elements sufficient for the transcription, translation,regulation, and if desired, sorting and secretion of the alteredvariable region polypeptides. The vectors can be suitable for expressionin either procaryotic or eukaryotic host systems so long as theexpression and regulatory elements function in the respective hostsystem. The expression vectors can additionally include regulatoryelements for inducible or cell type-specific expression. One skilled inthe art will know which host systems are compatible with a particularvector and which regulatory or functional elements are sufficient toachieve expression of the polypeptides in soluble, secreted or cellsurface forms.

[0126] Appropriate host cells, include for example, bacteria andcorresponding bacteriophage expression systems, yeast, avian, insect andmammalian cells. Methods for recombinant expression, screening andpurification of populations of altered variable regions or alteredvariable region polypeptides within such populations in various hostsystems are well known in the art and are described, for example, inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1992) and in Ausubel et al., CurrentProtocols in Molecular Biology, (Supplement 54), John Wiley & Sons, NewYork (2001). The choice of a particular vector and host system forexpression and screening of altered variable regions are known to thoseskilled in the art and will depend on the preference of the user.Moreover, expression of diverse populations of hetereomeric receptors ineither soluble or cell surface form using filamentous bacteriophagevector/host systems is well known in the art and is the subject matterof U.S. Pat. No. 5,871,974.

[0127] The expressed population of altered variable region polypeptidescan be screened for the identification of one or more altered variableregion species exhibiting optimized binding activity, for example,binding affinity substantially the same or greater than the donor CDRvariable region. Screening can be accomplished using various methodswell known in the art for determining the binding affinity of apolypeptide or compound. Additionaly, methods based on determining therelative affinity of binding molecules to their partner by comparing theamount of binding between the altered variable region polypeptides andthe donor CDR variable region can similarly be used for theidentification of species exhibiting binding affinity substantially thesame or greater than the donor CDR variable region. All of such methodscan be performed, for example, in solution or in solid phase. Moreover,various formats of binding assays are well known in the art and include,for example, immobilization to filters such as nylon or nitrocellulose;two-dimensional arrays, enzyme linked immunosorbant assay (ELISA),radioimmunoassay (RIA), panning and plasmon resonance. Such methods canbe found described in, for example, Harlow and Lane, supra, 1988.

[0128] For the screening of populations of polypeptides such as thealtered variable region populations produced by the methods of theinvention, immobilization of the populations of altered variable regionsto filters or other solid substrate can be advantageous because largenumbers of different species can be efficiently screened for antigenbinding. Such filter lifts allow for the identification of alteredvariable regions that exhibit substantially the same or greater bindingaffinity compared to the donor CDR variable region. Alternatively, ifthe populations of altered variable regions are expressed on the surfaceof a cell or bacteriophage, panning on immobilized antigen can be usedto efficiently screen for variants having antigen binding activity or todetermine the relative binding affinity of species within thepopulation.

[0129] Another affinity method for screening populations of alteredvariable regions polypeptides is a capture lift assay that is useful foridentifying a binding molecule having selective affinity for a ligand(Watkins et. al., (1997); WO 99/06834). This method employs theselective immobilization of altered variable regions to a solid supportand then screening of the selectively immobilized altered variableregions for selective binding interactions against the cognate antigenor binding partner. Selective immobilization functions to increase thesensitivity of the binding interaction being measured since initialimmobilization of a population of altered variable regions onto a solidsupport reduces non-specific binding interactions with irrelevantmolecules or contaminants which can be present in the reaction.

[0130] Another method for screening populations or for measuring theaffinity of individual altered variable region polypeptides is throughsurface plasmon resonance (SPR). This method is based on the phenomenonwhich occurs when surface plasmon waves are excited at a metal/liquidinterface. Light is directed at, and reflected from, the side of thesurface not in contact with sample, and SPR causes a reduction in thereflected light intensity at a specific combination of angle andwavelength. Biomolecular binding events cause changes in the refractiveindex at the surface layer, which are detected as changes in the SPRsignal. The binding event can be either binding association ordisassociation between a receptor-ligand pair. The changes in refractiveindex can be measured essentially instantaneously and therefore allowsfor determination of the individual components of an affinity constant.More specifically, the method enables accurate measurements ofassociation rates (k_(on),) and disassociation rates (k_(off)).

[0131] Measurements of k_(on), and k_(off) values can be used identifyaltered variable regions or optimized variable regions that aretherapeutically more efficacious. For example, an altered variableregion, or heteromeric binding fragment thereof, can be more efficaciousbecause it has, for example, a higher k_(on), valued compared tovariable regions and heteromeric binding fragments that exhibit similarbinding affinity. Increased efficacy is conferred because molecules withhigher k_(on) values can specifically bind and inhibit their target at afaster rate. Similarly, a molecule of the invention can be moreefficacious because it exhibits a lower k_(off) value compared tomolecules having similar binding affinity. Increased efficacy observedwith molecules having lower k_(off) rates can be observed because, oncebound, the molecules are slower to dissociate from their target.Although described with reference to the altered variable regions andoptimized variable regions of the invention, the methods described abovefor measuring association and dissociation rates are applicable toessentially any antibody or fragment thereof for identifying moreeffective binders for therapeutic or diagnostic purposes.

[0132] Methods for measuring the affinity, including association anddissociation rates using surface plasmon resonance are well known in theart and can be found described in, for example, Jönsson and Malmquist,Advances in Biosensors, 2:291-336 (1992) and Wu et al. Proc. Natl. Acad.Sci. USA, 95:6037-6042 (1998). Moreover, one apparatus well known in theart for measuring binding interactions is a BIAcore 2000 instrumentwhich is commercially available through Pharmacia Biosensor, (Uppsala,Sweden).

[0133] Using any of the above described screening methods, as well asothers well known in the art, an altered variable region havingoptimized binding activity, for example, binding affinity substantiallythe same or greater than the donor CDR variable region is identified bydetecting the binding of at least one altered variable region within thepopulation to its antigen or cognate ligand. In addition to optimizingfor antigen binding activity, catalytic activity can also be included inan invention antibody and optimized using the methods disclosed hereinfor binding affinity optimization. Accordingly, the above methods can bemodified to include the addition of substrate and reactants to screenfor optimized catalytic activity. Comparison, either independently orsimultaneously in the same screen, with the donor variable region willidentify those binders that have substantially the same or greaterbinding affinity as the donor. Those skilled in the art will know, orcan determine using the donor variable region, binding conditions whichare sufficient to identify selective interactions over non-specificbinding.

[0134] Detection methods for identification of binding species withinthe population of altered variable regions can be direct or indirect andcan include, for example, the measurement of light emission,radioisotopes, calorimetric dyes and fluorochromes. Direct detectionincludes methods that function without intermediates or secondarymeasuring procedures to assess the amount of bound antigen or ligand.Such methods generally employ ligands that are themselves labeled with adetectable moiety, for example, a radioactive, light emitting,fluorescent, calorimetric or enzyme moiety. In contrast, indirectdetection includes methods that function through an intermediate orsecondary measuring procedure. These methods generally employ moleculesthat specifically react with the antigen or ligand and can themselves bedirectly labeled with a detectable moiety or detected by a secondaryreagent. For example, an antibody specific for a ligand can be detectedusing a secondary antibody capable of interacting with the firstantibody specific for the ligand, again using the detection methodsdescribed above for direct detection. Moreover, for the specific exampleof screening for catalytic antibodies, the disappearance of a substrateor the appearance of a product can be used as an indirect measure ofbinding affinity or catalytic activity.

[0135] Isolated variable regions exhibit binding affinity as singlechains, in the absence of assembly into a heteromeric structure withtheir respective V_(H) or V_(L) subunits. As such, populations of V_(H)and V_(L) altered variable regions polypeptides can be expressed aloneand screened for binding activity, for example, optimized activityhaving substantially the same or greater binding affinity compared tothe CDR donor V_(H) or V_(L) variable region. Alternatively, populationsof V_(H) and V_(L) altered variable regions polypeptides can becoexpressed so that they self-assemble into heteromeric altered variableregion binding fragments. The heteromeric binding fragment populationcan then be screened for species exhibiting binding affinitysubstantially the same or greater than the CDR donor variable regionbinding fragment.

[0136] Employing the methods for simultaneously grafting and optimizing,or for optimizing, it is possible to generate heteromeric variableregion binding fragments having increases in affinities of greater thanabout 2-fold, 3-fold, 4-fold, 5-fold, 8-fold or 10-fold. In particular,heteromeric variable region binding fragments can be generated havingincreases in affinities of greater than 12-fold, 15-fold, 20-fold, and25-fold as well as affinities greater than 50-fold, 100-fold, 200-fold,500-fold or 1000-fold compared to the donor or parent molecule.

[0137] Additionally, the methods described herein for optimizing arealso are applicable for producing catalytic heteromeric variable regionfragments or for optimizing their catalytic activity. Catalytic activitycan be optimized by changing, for example, the on or off rate ofsubstrate binding, the substrate binding affinity, the transition statebinding affinity, the turnover rate (kcat) or the Km. Methods formeasuring these characteristics are well known in the art (see, forexample Segel, Enzyme Kinetics, John Wiley & Sons, New York (1975)).Such methods can be employed in the screening steps of the methodsdescribed above when used for optimizing the catalytic activity of aheteromeric variable region binding fragment.

[0138] Additionally, the methods for conferring donor CDR bindingaffinity onto an antibody acceptor variable region framework areapplicable for grafting CDRs as described by Kabat et al., supra,Chothia et al., supra or MacCallum et al., supra. The methods similarlycan be used for grafting into an acceptor framework overlapping regionsor combinations of CDRs as described in Kabat et al., supra, Chothia etal., supra or MacCallum et al., supra. Generally, variable region CDRsare grafted by identifying the boundries described by one of the CDRdefinitions known in the art and set forth herein. However, because themethods are directed to constructing and screening populations of CDRgrafted altered variable regions, which can incorporate relevant aminoacid position changes in both the framework and CDR regions, and suchvariations can, for example, compensate or augment amino acid changeselsewhere in the variable region, the exact boundry of a particular CDRor set of variable region CDRs can be varied. Therefore, the exact CDRregion to graft, whether it is the region described by Kabat et al.,Chothia et al. or MacCallum et al., or any combination thereof, willessentially depend on the preference of the user.

[0139] Similarly, the methods described previously for optimizing thebinding affinity of an antibody also are applicable for use withessentially any variable region for which an encoding nucleic acid is,or can be made, available. As with the methods for conferring donor CDRbinding affinity, many applications of the methods for optimizingbinding affinity will be for modifying the binding affinity of CDRgrafted variable regions having human frameworks. Again, such moleculesare significantly less antigenic in human patients and thereforetherapeutically valuable in the treatment of human diseases. However,the methods of the invention for optimizing the binding affinity of avariable region are applicable to all species of variable regions.Therefore, the invention includes binding affinity optimization ofvariable regions derived from human, mouse, rat, rabbit, goat andchicken, or any other desired species.

[0140] The methods of the invention have been described with referenceto variable regions and heteromeric variable region binding fragments.Those skilled in the art will understand that all of such methods areapplicable to whole antibodies and functional fragments thereof as wellas to regions and functional domains other than the antigen bindingvariable region of antibodies, if desired.

[0141] An association rate can be determined in any non-equilibriummixture including, for example, one formed by rapidly contacting abinding polypeptide and ligand or by rapidly changing temperature. Anon-equilibrium mixture can be a pre-equilibrium mixture. Apre-equilibrium mixture can be formed, for example, by contacting asoluble binding polypeptide and soluble ligand in a condition where theamount of total ligand and total binding polypeptide in the detectionchamber are constant. Measurements of association rates inpre-equilibrium mixtures can be made in formats providing rapid mixingof binding polypeptide with ligand and rapid detection of changingproperties of the binding polypeptide or ligand on a timescale ofmilliseconds or faster. Stopped flow and rapid quench flow instrumentssuch as those described below provide a convenient means to measurenon-equilibrium kinetics. The association rate can also be measured innon-equilibrium mixtures including, for example, solutions containinginsoluble species of binding polypeptide, ligand or binding polypeptidebound to ligand, or solutions containing variable concentrations oftotal ligand or total binding polypeptide. Measurement of an associationrate in a non-equilibrium mixture can be made in formats providingattachment of a ligand to a surface and continuous flow of a solutioncontaining the binding polypeptide over the surface, or vice-versa,combined with rapid detection of changing properties of the bindingpolypeptide, ligand or surface such that measurements are made on atimescale of milliseconds or faster. Examples of formats providingnon-equilibrium measurement of association rates include surface plasmonresonance instruments and evanescent wave instruments.

[0142] Association rate measurements can be made by detecting the changein a property of the binding polypeptide or ligand that exists betweenthe bound and unbound state or by detecting a change in the surroundingenvironment when binding polypeptide and ligand associate. Properties ofthe binding polypeptide or ligand that can change upon association andthat can be used to measure association rates include, for example,absorption and emission of heat, absorption and emission ofelectromagnetic radiation, affinity for a receptor, molecular weight,density, mass, electric charge, conductivity, magnetic moment of nuclei,spin state of electrons, polarity, molecular shape, or molecular size.Properties of the surrounding environment that can change when bindingpolypeptide associates with ligand include, for example, temperature andrefractive index of surrounding solvent.

[0143] Formats for measuring association rates in pre-equilibriummixtures include, for example, stopped flow kinetic instruments andrapid quench flow instruments. A stopped flow instrument can be used topush solutions containing a binding polypeptide and ligand from separatereservoirs into a mixing chamber just prior to passage into a detectioncell. The instrument can then detect a change in one or more of theabove described properties to monitor progress of the binding event. Arapid quench flow instrument can be used to rapidly mix a solutioncontaining a binding polypeptide with a solution containing a ligandfollowed by quenching the binding reaction after a finite amount oftime. A change in one or more of the above described properties can thenbe detected for quenched mixtures produced by quenching at differenttimes following mixing. Quenching can be performed for example byfreezing or addition of a chemical quenching agent so long as thequenching step does not inhibit detection of the property relied uponfor measurement of binding rate. Thus, a rapid quench instrument can beuseful, for example, in situations where spectroscopic detection is notconvenient. A variety of instruments are commercially available fromvendors such as KinTek Corp. (State College, Pa.) and Hi-Tech Scientific(Salisbury, UK).

[0144] Formats for measuring association rates in non-equilibriummixtures include, for example, surface plasmon resonance and evanescentwave instruments. Surface plasmon resonance and evanescent wavetechnology utilize a ligand or binding polypeptide attached to abiosensor surface and a solution containing either the bindingpolypeptide or ligand respectively that is passed over the biosensorsurface. The change in refractive index of the solution that occurs atthe surface of a chip when binding polypeptide associates with ligandcan be measured in a time dependent fashion. For example, surfaceplasmon resonance is based on the phenomenon which occurs when surfaceplasmon waves are excited at a metal/liquid interface. Light is directedat, and reflected from, the side of the surface not in contact withsample, and SPR causes a reduction in the reflected light intensity at aspecific combination of angle and wavelength. Biomolecular bindingevents cause changes in the refractive index at the surface layer, whichare detected as changes in the SPR signal. The binding event can beeither binding association or disassociation between a receptor-ligandpair. The changes in refractive index can be measured essentiallyinstantaneously and therefore allows for determination of the individualcomponents of an affinity constant. More specifically, the methodenables accurate measurements of association rates (k_(on)) anddisassociation rates (k_(off)). Surface plasmon resonance instrumentsare available in the art including, for example, the BIAcore instrument,IBIS system, SPR-CELLIA system, Spreeta, and Plasmon SPR and evanescentwave technology is available in the Iasys system as described, forexample, in Rich and Myszka, Curr. Opin. Biotech. 11:54-61 (2000).

[0145] Another method for measuring binding affinity includescomparative ELISA. As disclosed herein, an approximation of changes inaffinity based on shifts in half-maximal binding was used to identifyk_(on) and k_(off) values relative to wild type (Example III). Such amethod is particularly useful for screening large numbers of variants,whereas the above-described methods can be used for detailed analysis ofbinding activity.

[0146] The association rate can be determined by measuring a change in aproperty of a ligand or binding polypeptide at one or more discreet timeintervals during the binding event using, for example, the methodsdescribed above. Measurements determined at discreet time intervalsduring the binding event can be used to determine a quantitative measureof association rate or a relative measure of association rate.Quantitative measures of association rate can include, for example, anassociation rate value or k_(on) value. Quantitative values ofassociation rate or k_(on) can be determined from a mathematical orgraphical analysis of a time dependent measurement. Such analyses arewell known in the art and include algorithms for fitting data to a sumof exponential or linear terms or algorithms for computer simulation tofit data to a binding model as described for example in Johnson, Cur.Opin. Biotech. 9:87-89 (1998), which is incorporated herein byreference.

[0147] Association rates can be determined from mixtures containinginsoluble species or variable concentrations of total ligand or totalbinding polypeptide using mathematical and graphical analyses such asthose described above if effects of mass transport are accounted for inthe reaction. One skilled in the art can account for mass transport bycomparing association rates under conditions having similar limitationswith respect to mass transport or by adjusting the calculatedassociation rate according to models available in the art including, forexample those described in Myszka et al., Biophys. J. 75:583-594 (1998),which is incorporated herein by reference.

[0148] A higher value of either the association rate or k_(on) isgenerally indicative of improved therapeutic potency. Thus, quantitativedeterminations provide an advantage by allowing comparison between anassociation rate of a binding polypeptide and a therapeutic controldetermined by different methods so long as the methods used areunderstood by one skilled in the art to yield consistent results.

[0149] A relative measure of association rate can include, for example,comparison of association rate for two or more binding polypeptidesbinding to ligand under similar conditions or comparison of associationrate for a binding polypeptide binding to ligand with a predefined rate.Comparison of association rate for two or more binding polypeptides caninclude a standard of known association rate or a molecule of knowntherapeutic effect. A predefined rate used for comparison can bedetermined by calibrating the measurement relative to a previouslymeasured rate including, for example, one available in the scientificliterature or in a database. An example of a comparison with apredefined rate is selection of the species of binding polypeptide boundto ligand at a discreet time interval defined by the predefined rate byusing a time actuated selection device.

[0150] For purposes of comparison, the association rate of a bindingpolypeptide and ligand can be determined relative to association ratefor a therapeutic control and the same ligand. A comparison can also bemade according to a quantitative association rate for bindingpolypeptide and ligand compared to a quantitative association rate for atherapeutic control and ligand. Relative or quantitative associationrates can be determined by the methods described above. Determination ofassociation rates for a binding polypeptide associating with a ligandcan be performed simultaneously with a binding polypeptide andtherapeutic control or at separate times, provided conditions aresufficiently similar in each assay to allow valid comparison. Thus,association rate determined for a binding polypeptide can be compared toa previously measured association rate for a therapeutic control.

[0151] A binding polypeptide having improved therapeutic potency can bedistinguished from a binding polypeptide that has an increased K_(a) fora ligand but not improved therapeutic potency. Methods for identifying atherapeutic binding polypeptide based on K_(a) rely on an equilibriummeasurement which, absent time dependent measurements made in anon-equilibrium condition, are inaccurate for identifying a bindingpolypeptide having increased association rate and therefore improvedtherapeutic potency. According to the relationship K_(a)=k_(on)/k_(off),an increased K_(a) for association of a binding polypeptide and ligandcan be due to changes in k_(on) or k_(off). For example, a bindingpolypeptide having improved therapeutic potency can have a reduced K_(a)if a reduction in k_(off) occurs that over compensates for an increasein k_(on). Thus, changes in K_(a), being influenced by changes ink_(off), do not unambiguously correlate with changes in therapeuticpotency since binding polypeptides having improved therapeutic potencycan display either reduced or increased K_(a).

[0152] For optimization of binding activity of an antibody of theinvention, the fold increase in association rate can be indicated by anincrease in k_(on). Therefore, k_(on) can be about 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more usingmethods described herein. The k_(on) can be at least about 1×10² M⁻¹s⁻¹,2×10² M⁻¹s⁻¹, 5×10² M⁻¹s⁻¹, 1×10³ M⁻¹s⁻¹, 2×10³ M⁻¹s⁻¹, 5×10³ M⁻¹s⁻¹,1×10⁴ M⁻¹s⁻¹, 2×10⁴ M⁻¹s⁻¹, 5×10⁴ M⁻¹s⁻¹, 1×10⁵ M⁻¹s⁻¹, 2×10⁵ M⁻¹s⁻¹, or3×10⁵ M⁻¹s⁻¹. The k_(on) can also be increased to at least about 5×10⁵M⁻¹s⁻¹, 7×10⁵ M⁻¹s⁻¹, 9×10 M⁻¹s⁻¹, 1×10⁶ M⁻¹s⁻¹, 3×10⁶M⁻¹s⁻¹, 5×10⁶M⁻¹s⁻¹, 7×10⁶ M⁻¹s⁻¹, 9×10⁶ M⁻¹s⁻¹ or 1×10⁷ M⁻¹s⁻¹ or more. Furthermore,the increase in k_(on) resulting in improved therapeutic potency can beindependent of an effect of a change in K_(a) for the bindingpolypeptide. The binding polypeptide having an increase in k_(on) canhave a K_(a) value similar to K_(a) for its parent polypeptide or aK_(a) value lower than K_(a) for its parent polypeptide.

[0153] The invention also provides nucleic acids encoding the antibodiesand CDRs of the invention. The invention further provides nucleic acidsencoding the mouse antibodies HUIV26 (SEQ ID NOS:1 and 3) and HUI77 (SEQID NOS:5 and 7) (see FIGS. 2 and 3). Further provided are nucleic acidsencoding HUIV26 CDRs (SEQ ID NOS:20, 22, 24, 26, 28 and 30) and encodingHUI77 CDRs (SEQ ID NOS:32, 34, 36, 38, 40 and 42). Such nucleic acidsinclude nucleic acids having degenerate codons encoding any or all ofthe amino acids in the CDRs. For example, the invention provides nucleicacids encoding HUIV26 CDRs: VL CDR1, SEQ ID NOS:19; V_(L) CDR2, SEQ IDNO:21; V_(L) CDR3, SEQ ID NO:23; V_(H) CDR1, SEQ ID NO:25; V_(H) CDR2,SEQ ID NO:27; and V_(H) CDR3, SEQ ID NO:29. The invention also providesnucleic acids encoding HUI77 CDRs: V_(L) CDR1, SEQ ID NOS:31; V_(L)CDR2, SEQ ID NO:33; V_(L) CDR3, SEQ ID NO:35; V_(H) CDR1, SEQ ID NO:37;V_(H) CDR2, SEQ ID NO:39; and V_(H) CDR3, SEQ ID NO:41. Also includedare degenerate versions of such nucleic acids such that they encode theamino acid sequences referenced as SEQ ID NOS:20, 22, 24, 26, 28 and 30for HUIV26 and SEQ ID NOS:32, 34, 36, 38, 40 and 42 for HUI77.

[0154] Further provided are nucleic acids encoding a HUIV26 or HUI77 CDRcontaining one or more amino acid subsitutions. For example, theinvention provides nucleic acids encoding the CDRs of HUIV26 and HUI77having single or multiple amino acid substitutions, as disclosed herein.If a nucleic acid encoding a CDR having one or more amino acidsubstitution is derived, for example, from one of SEQ ID NOS:19, 21, 23,25, 27 or 29 for HUIV26 or SEQ ID NOS:31, 33, 35, 37, 39 or 41 forHUI77, the amino acid substitutions can be encoded by any of thecorresponding degenerate codons for that amino acid. Nucleic acidsencoding such CDR variants can also include degenerate codons at any orall of the wild type amino acid positions.

[0155] Throughout the application, various nucleic acids andoligonucleotide primers, in addition to the naturally occurringnucleotides A, C, G, T or U, refer to standard abbreviations: R=G or A;Y=T/U or C; M=A or C; K=G or T/U; S=G or C; W=A or T/U; B=G, C or T/U;D=A, G or T/U; H=A, C or T/U; V=A, G or C; N=any nucleotide.

[0156] The antibodies of the invention have binding activity for acryptic collagen epitope. The HUIV26 and HUI77 antibodies have beenshown to target to angiogenic vasculature (see Xu et al., supra, 2001;WO 00/40597). Accordingly, the grafted HUIV26 and HUI77 antibodies ofthe invention, which specifically bind to a cryptic collagen epitope,similarly can target to angiogenic vasculature. One of the mostsignificant and important aspects of the monoclonal antibodies HUIV26and HUI77, and the grafted forms thereof disclosed herein, is that oftheir specificity. It is expected that systemic administration ofantibodies of the invention will have minimal if any toxic side effectssince the cryptic epitope(s) that is recognized by the HUIV26 and HUI77antibodies is/are not exposed in mature native triple helical collagenbut is only exposed upon denaturaion, for example, heat denaturation orproteolytic denaturation. Thus, little, if any, binding under normalphysiological conditions is expected.

[0157] Moreover, the cryptic collagen domain(s) to which HUIV26 andHUI77 bind represents a novel therapeutic target for the treatment ofnumerous neovascular diseases including tumor growth and metastasis,diabetic retinopathy and other related ocular diseases such as maculardegeneration, psoriasis, and rheumatoid arthritis. Other exemplarydiseases associated with angiogenesis include, but are not limited to,inflammatory disorders such as immune and non-immune inflammation,chronic articular rheumatism and psoriasis, disorders associated withinappropriate or inopportune invasion of vessels such as diabeticretinopathy, neovascular glaucoma, restenosis, capillary proliferationin atherosclerotic plaques and osteoporosis, and cancer associateddisorders, such as solid tumors, solid tumor metastases, angiofibromas,retrolental fibroplasia, hemangiomas, Kaposi's sarcoma and the likecancers which require neovascularization to support tumor growth. Otherexemplary tumors include melanoma, carcinoma, sarcoma, fibrosarcoma,glioma and astrocytoma, and the like.

[0158] Thus, the methods of the invention can be used to treat anindividual having a disease associated with angiogenesis, includingthose described above. The methods can be used to ameliorate a sign orsymptom associated with a disease. For example, in the case of cancertreatment, the methods can be used to inhibit tumor growth. One skilledin the art will know or can readily determine an appropriate sign orsymptom associated with a disease suitable for determining theeffectiveness of a therapeutic application using an antibody of theinvention.

[0159] The antibodies of the invention can also be used as an importantdiagnostic and imaging reagent for the early detection of aberrantneovascularization associated with invasive tumor growth and metastasis.The antibodies of the invention can also be used in staging and gradingof tumors since invasive tumor in contrast to benign lesions are likelyto be associated with degradation of the surrounding basement membrane.

[0160] Thus, the invention provides a method of targeting angiogenicvasculature, comprising administering an antibody, or functionalfragment thereof, the antibody or functional fragment thereof havingspecific binding activity for a cryptic collagen epitope, wherein theantibody or functional fragment is an antibody of the invention. Forexample, the antibodies can comprise one or more CDRs, including wildtype CDRs or variants thereof, of the HUIV26 and HUI77 antibodies, asdisclosed herein. The methods of targeting angiogenic vasculature can beused for therapeutic and/or diagnostic purposes.

[0161] For therapeutic purposes, the antibody, or functional fragmentthereof, can be administered as a therapeutic agent itself or canfurther comprise a therapeutic moiety. In the case of a therapeuticmoiety, the moiety can be a drug such as a chemotherapeutic agent,cytotoxic agent, toxin, or anti-angiogenic agent, which refers to amolecule that reduces or inhibits angiogenesis. For example, a cytotoxicagent can be a radionuclide or chemical compound. Exemplaryradionuclides useful as therapeutic agents include, for example, X-rayor y-ray emitters. In addition, a moiety can be a drug delivery vehiclesuch as a chambered microdevice, a cell, a liposome or a virus, whichcan contain an agent such as a drug or a nucleic acid.

[0162] Exemplary therapeutic agents include, for example, theanthracyclin, doxorubicin, which has been linked to antibodies and theantibody/doxorubicin conjugates have been therapeutically effective intreating tumors (Sivam et al., Cancer Res. 55:2352-2356 (1995); Lau etal., Bioorg. Med. Chem. 3:1299-1304 (1995); Shih et al., Cancer Immunol.Immunother. 38:92-98 (1994)). Similarly, other anthracyclins, includingidarubicin and daunorubicin, have been chemically conjugated toantibodies, which have delivered effective doses of the agents to tumors(Rowland et al., Cancer Immunol. Immunother. 37:195-202 (1993);Aboud-Pirak et al., Biochem. Pharmacol. 38:641-648 (1989)).

[0163] In addition to the anthracyclins, alkylating agents such asmelphalan and chlorambucil have been linked to antibodies to producetherapeutically effective conjugates (Rowland et al., Cancer Immunol.Immunother. 37:195-202 (1993); Smyth et al., Immunol. Cell Biol.65:315-321 (1987)), as have vinca alkaloids such as vindesine andvinblastine (Aboud-Pirak et al., supra, 1989; Starling et al., Bioconj.Chem. 3:315-322 (1992)). Similarly, conjugates of antibodies andantimetabolites such as 5-fluorouracil, 5-fluorouridine and derivativesthereof have been effective in treating tumors (Krauer et al., CancerRes. 52:132-137 (1992); Henn et al., J. Med. Chem. 36:1570-1579 (1993)).Other chemotherapeutic agents, including cis-platinum (Schechter et al.,Int. J. Cancer 48:167-172 (1991)), methotrexate (Shawler et al., J.Biol. Resp. Mod. 7:608-618 (1988); Fitzpatrick and Garnett, AnticancerDrug Des. 10:11-24 (1995)) and mitomycin-C (Dillman et al., Mol.Biother. 1:250-255 (1989)) also are therapeutically effective whenadministered as conjugates with various different antibodies. Atherapeutic agent can also be a toxin such as ricin.

[0164] A therapeutic agent can also be a physical, chemical orbiological material such as a liposome, microcapsule, micropump or otherchambered microdevice, which can be used, for example, as a drugdelivery system. Generally, such microdevices, should be nontoxic and,if desired, biodegradable. Various moieties, including microcapsules,which can contain an agent, and methods for linking a moiety, includinga chambered microdevice, to an antibody of the invention are well knownin the art and commercially available (see, for example, “Remington'sPharmaceutical Sciences” 18th ed. (Mack Publishing Co. 1990), chapters89-91; Harlow and Lane, Antibodies: A laboratory manual (Cold SpringHarbor Laboratory Press 1988)).

[0165] For diagnostic purposes the antibody, or functional fragmentthereof, can further comprise a detectable moiety. A detectable moietycan be, for example, a radionuclide, fluorescent, magnetic, calorimetricmoeity, and the like. For in vivo diagnostic purposes, a moiety such asa gamma ray emitting radionuclide, for example, indium-ill ortechnitium-99, can be linked to an antibody of the invention and,following administration to a subject, can be detected using a solidscintillation detector. Similarly, a positron emitting radionuclide suchas carbon-11 or a paramagnetic spin label such as carbon-13 can belinked to the molecule and, following administration to a subject, thelocalization of the moiety can be detected using positron emissiontransaxial tomography or magnetic resonance imaging, respectively. Suchmethods can identify a primary tumor as well as a metastatic lesion.

[0166] For diagnostic purposes, the antibodies of the invention can beused to determine the levels of denatured collagen in a tissue or in abodily fluid. The level of denatured collagen can be determined in atissue sample obtained from an individual, for example, by tissuebiopsy. Exemplary bodily fluids include, but are not limited to, serum,plasma, urine, synovial fluid, and the like.

[0167] The invention also provides a method of inhibiting angiogenesisby administering an antibody, or functional fragment thereof, where theantibody or functional fragment thereof has specific binding activityfor a cryptic collagen epitope, where the antibody comprises one or moreCDRs of the invention. For example, an antibody of the invention can beadministered so that angiogenesis is inhibited in a tissue of anindividual. The invention further provides a method of targeting a tumorby administering an invention antibody. The invention also provides amethod of inhibiting tumor growth by administering an antibody, orfunctional fragment thereof, of the invention.

[0168] The antibodies of the invention can also be used for in vivo orin vitro diagnostic applications. Thus, the invention provides a methodof detecting angiogenic vasculature by contacting angiogenic vasculaturewith an antibody, or functional fragment thereof, of the invention.Angiogenic vasculature can be imaged in vivo by administering anantibody of the invention, either alone or attached to a detectablemoiety, to an individual. The angiogenic vasculature can thus bedetected in vivo. Alternatively, the antibody can be administered to atissue obtained from an individual, for example, a tissue biopsy, suchthat an antibody of the invention can be used in vitro for diagnosticpurposes to detect angiogenic vasculature.

[0169] A therapeutic or detectable moiety can be coupled to an antibodyof the invention, or functional fragment thereof, by any of a number ofwell known methods for coupling or conjugating moieties. It isunderstood that such coupling methods allow the attachment of atherapeutic or detectable moiety without interfering or inhibiting thebinding activity of the antibody, that is, the ability to bind a crypticcollagen site. Methods for conjugating moieties to an antibody of theinvention, or functional fragment thereof, are well known to thoseskilled in the art (see, for example, Hermanson, BioconjugateTechniques, Academic Press, San Diego (1996)).

[0170] When administered to a subject, the antibody of the invention isadministered as a pharmaceutical composition containing, for example,the antibody and a pharmaceutically acceptable carrier. As disclosedherein, the antibody can be coupled to a therapeutic or detectablemoiety. Pharmaceutically acceptable carriers are well known in the artand include, for example, aqueous solutions such as water orphysiologically buffered saline or other solvents or vehicles such asglycols, glycerol, oils such as olive oil or injectable organic esters.

[0171] A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize or to increasethe absorption of the conjugate. Such physiologically acceptablecompounds include, for example, carbohydrates, such as glucose, sucroseor dextrans, antioxidants, such as ascorbic acid or glutathione,chelating agents, low molecular weight proteins or other stabilizers orexcipients. One skilled in the art will know that the choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on the route ofadministration of the composition. The pharmaceutical composition alsocan contain an agent such as a cancer therapeutic agent.

[0172] One skilled in the art will know that a pharmaceuticalcomposition containing an antibody of the invention can be administeredto a subject by various routes including, for example, orally orparenterally, such as intravenously. The composition can be administeredby injection or by intubation. The pharmaceutical composition also canbe an antibody linked to liposomes or other polymer matrices, which canhave incorporated therein, for example, a drug such as achemotherapeutic agent (Gregoriadis, Liposome Technology, Vols. I toIII, 2nd ed. (CRC Press, Boca Raton Fla. (1993), which is incorporatedherein by reference). Liposomes, for example, which consist ofphospholipids or other lipids, are nontoxic, physiologically acceptableand metabolizable carriers that are relatively simple to make andadminister.

[0173] For diagnostic or therapeutic methods disclosed herein, aneffective amount of the antibody and therapeutic moiety is administeredto the subject. As used herein, the term “effective amount” means theamount of the pharmaceutical composition that produces the desiredeffect. An effective amount often will depend on whether the antibodyitself is administered or whether the antibody is linked to a moiety andthe type of moiety. Thus, a lesser amount of a radiolabeled molecule canbe required for imaging as compared to the amount of a radioactivedrug/antibody conjugate administered for therapeutic purposes. Aneffective amount of a particular antibody/moiety for a specific purposecan be determined using methods well known to those in the art. Oneskilled in the art can readily determine an appropriate dose of anantibody of the invention for an effective amount for therapeutic ordiagnostic purposes.

[0174] For therapeutic or in vivo diagnostic purposes, it is understoodthat any of a variety of methods of administration can be used so longas the administration is effective for a desired purpose. Such methodsof administration include, for example, intravenous, transdermal,intrasynovial, intramuscular, intratumoral, intraocular, intranasal,intrathecal, topical, oral, or the like. One skilled in the art canreadily determine an appropriate mode of administration depending on thedesired therapeutic effect or desired diagnostic purpose.

[0175] Furthermore, it is understood that for therapeutic or diagnosticapplications, an antibody of the invention in general is administered toa mammal, for example, a human. Applications of an antibody of theinvention for domestic animals or agricultural purposes include othermammals, for example, a non-human primate, pig, cow, horse, goat, sheep,mule, donkey, dog, cat, rabbit, mouse, rat, and the like.

[0176] It is understood that any of the therapeutic methods disclosedherein using an antibody of the invention can be used in combinationwith other therapeutic methods. For example, an antibody of theinvention, either the antibody itself or an antibody attached to atherapeutic agent, can be administered simultaneously or sequentiallywith other therapeutic treatment regimens. For example, an antibody ofthe invention can be administered alone or in combination with anothertherapeutic treatment, including any of the therapeutic drugs disclosedherein as well as other drugs well known to those skilled in the art fortreating a particular disease. For example, in the case of treating acancer, an antibody of the invention can be administered simultaneouslyor sequentially with another chemotherapeutic agent such as a drug orradionuclide. Similarly, an antibody of the invention can be combinedwith other treatment regimens such as surgery by administering theantibody before, during or after surgery. One skilled in the art willknow or can readily determine a desirable therapeutic treatment to beused in combination with an antibody of the invention, as desired. Thus,an antibody of the invention can be administered in conjunction withother therapeutic regimens, including but not limited to chemotherapy,radiation therapy, surgery, and the like.

[0177] The invention additionally provides a method of inhibitingmetastasis using an antibody of the invention. The method can includethe step of administering an antibody, or functional fragment thereof,having binding activity for a cryptic collagen epitope. The antibody canbe, for example, an antibody comprising one or more CDRs having a leastone amino acid substitution in one or more heavy or light chain CDRs ofantibodies HUIV26 and HUI77. As used herein, inhibiting metastasisrefers to decreasing the number and/or size of metastatic sites remotefrom a primary tumor site. The method of inhibiting metastasis caninvolve using an antibody of the invention that blocks adhesion of tumorcells to a cryptic collagen epitope that is exposed after remodeling oftissues by the action of collagen-degrading enzymes secreted by tumorcells.

[0178] As disclosed herein, a variant of HUI77 having one or more aminoacid substitutions in one or more CDRs inhibited proliferation ofmelanoma cells in vitro (see Example VI). An antibody of the inventioncan block access to or inhibit binding of a survival or proliferativesignal delivered to a tumor cell. Thus, the invention also provides amethod of targeting a tumor cell by administration of an antibody of theinvention having binding activity for a cryptic collagen epitope thatblocks access to a survival or proliferative signal delivered to thetumor cell by a cryptic collagen site.

[0179] For methods of inhibiting angiogenesis, the angiogenicvasculature can be associated with a tumor. The methods of the inventioncan also be used to inhibit tumor growth directly, alone or incombination with inhibiting angiogenic vasculature of the tumor. Themethods of the invention can additionally be used to inhibit metastasis,alone or in combination with inhibiting tumor angiogenic vasculatureand/or tumor growth. Exemplary tumors include, but are not limited to,those disclosed herein, including melanoma, carcinoma, sarcoma,fibrosacroma, glioma, astrocytoma, and the like. Methods for testing theeffect a HUIV26 or HUI77 variant for inhibition of angiogenesis orinhibition of tumor growth can be performed as described previouslyusing, for example, assays such as the rat corneal micropocketangiogenesis assay, chick embryo tumor growth assay, or SCID mouse tumorgrowth assay, as described in Xu et al., supra, 2001, or any other wellknown assays for measuring inhibition of angiogenesis, inhibition oftumor growth, or inhibition of metastasis.

[0180] The methods of the invention can also be applied to inhibitingnon-tumor angiogenic vasculature. Such applications to non-tumorangiogenic vasculature can include tissue that is inflamed and in whichangiogenesis is occurring. Exemplary non-tumor diseases associated withangiogenic vasculature suitable for treatment with an antibody of theinvention include, but are not limited to, those disclosed herein,including arthritis, ocular disease, retinal disease, hemangioma, andthe like. The antibodies of the invention can also be used to inhibitpsoriasis, macular degeneration, restenosis, and the like, or any tumoror non-tumor disease associated with increased accessibility of acryptic collagen epitope for which an antibody of the invention hasbinding activity.

[0181] It is understood that modifications which do not substantiallyaffect the activity of the various embodiments of this invention arealso provided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Cloning of Heavy and Light Chain Variable Regions of HUIV26and HU177 Antibodies

[0182] This example describes the cloning of HUIV26 and HUI77 antibodyvariable regions.

[0183] The variable regions of the HUIV26 and HUI77 antibodies werecloned from hybridomas expressing these mouse monoclonal antibodies andsequenced. Briefly, total mRNA was isolated from the respective mousehybridoma cells using Oligotex® Direct mRNA Micro kit (Qiagen; ValenciaCalif.). First strand cDNA was synthesized from the mRNA usingSuperScript Preamplification System (GibcoBRL/Invitrogen; CarlsbadCalif.). Antibody variable region sequences were amplified by PCR usinga set of 5′ primers designed for signal sequences of mouse light chainsor heavy chains to pair with single 3′ primer to mouse kappa chainconstant region for V_(L) or IgM CH1 region for V_(H) sequences. Thesequences of the 5′ primers for the signal peptide of mouse antibodyheavy and light chain as well as constant region primers are shown inFIG. 1. The 3′ primer for mouse kappa light chain constant region(primer 2650; SEQ ID NO:212) corresponds to amino acids 115-123. The 3′primer for mouse IgM CH1 region (primer 2656; SEQ ID NO:213) correspondsto amino acids 121-114. The 3′ primer for mouse IgM CH1 region (primer2706; SEQ ID NO:214) corresponds to amino acids 131-124.

[0184] The DNA fragments were isolated from PCR reactions, with a mainproduct of about 400 bp in length. The DNA fragments were cloned intothe pCR2.1 vector. The inserted DNA fragments were sequenced with bothforward and reversed M13 primers. The DNA sequences were compared withan antibody sequence database. The N-terminal amino acid sequence of theHUIV26 and HUI77 antibodies were determined, and the sequences of theDNA fragments were also compared to the N-terminal amino acid sequencesof the corresponding antibody.

[0185] The HUIV26 V_(L) encoding nucleic acid was cloned with 5′ primermK2 (primer 2664; SEQ ID NO:185) and 3′ primer 2650 (SES ID NO:212). Apartial sequence of HUIV25 V_(L) is ATCTTCTTGCTGTTCTGGGTATCTGGAACCTGTGGG(SEQ ID NO:215), with the MK2 primer underlined and the partial sequencecoding for mouse signal peptide in italics. The HUIV26 V_(H) encodingnucleic acid was cloned with 5′ primer MH12 (primer 2731; SEQ ID NO:203)and 3′ primer 2706 (SEQ ID NO:214).

[0186] The HUI77 V_(L) encoding nucleic acid was cloned with 5′ primermK1 (primer 2663; SEQ ID NO:184) and 3′ primer 2650 (SEQ ID NO:212). Apartial sequence of HUI77 V_(L) is TTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGT(SEQ ID NO:216), with the mK1 primer underlined and the partial sequencecoding for mouse signal peptide in italics. The HUI77 encoding nucleicacid was cloned with 5′ primers MH15 (primer 2734; SEQ ID NO:206) orMH16 (primer 2735; SEQ ID NO:207) and 3′ primer 2656 (SEQ ID NO:213).

[0187] The sequences of the heavy and light chain nucleotide and aminoacid sequences for HUIV26 and HUI77 are shown in FIGS. 2 and 3,respectively. Using the numbering system of Kabat, supra, the CDRs ofthe heavy and light chains were identified for each of the HUIV26 andHUI77 antibodies (underlined in FIGS. 2C and 3C).

[0188] An alignment of the HUI77 V_(L) nucleotide sequence (SEQ ID NO:9)with the nucleotide sequence of the human framework fusion DPK13/JK1(SEQ ID NO:17) is shown in FIG. 3D. The corresponding light chain aminoacid sequences are referenced as SEQ ID NO:10 and SEQ ID NO:18 for HUI77and DPK13/JK1, respectively.

[0189] This example describes the cloning and the sequence of mouseantibodies HUIV26 and HUI77.

EXAMPLE II Generation of CDR Variant Libraries of HUIV26 and HUI77Antibodies

[0190] This example describes the generation of CDR variant libraries ofHUIV26 and HUI77 antibodies for CDR optimization.

[0191] The CDR3 regions of antibodies HUIV26 and HUI77 were optimized bygenerating a library of CDR variants. Primers for light chain CDR3 andheavy chain CDR3 were used to generate a library of CDR3 variants, wherethe primer was synthesized to encode more than one amino acid one ormore positions in CDR3. Following synthesis of primers encoding CDR3variants, the variant CDR3 regions were assembled into light chain(V_(L)) and heavy chain (V_(H)) regions.

[0192] Briefly, humanized V_(L) and V_(H) genes of HUI77 and HUIV26antibodies were assembled with the primers shown in FIGS. 4A and 5A,respectively, using PCR or primer-elongation-ligantion. Variable regiongenes containing CDR3 mutations were assembled by replacing the wildtype CDR3 primer (IV26-17, IV26-h7, 177-17 or 177-h7) with the group ofmutant primers corresponding to that CDR. The assembled variable regionswere then amplified and asymmetrically biotinylated on plus strand byPCR using primers B-pelB and 224 for V_(L) and B-phA and 1200a for H_(V)genes. The primers for amplification of humanized V_(L) and V_(H)sequences and the isolation of minus strand DNA were: B-pelB, Biotin-TTACTC GCT GCC CAA CCA GCC ATG GCC (SEQ ID NO:220); 224, GAC AGA TGG TGCAGC CAC AGT (SEQ ID NO:221); B-phoA, Biotin-TTA CTG TTT ACC CCT GTG ACAAAA GCC (SEQ ID NO:222); and 1200a, GAA GAC CGA TGG GCC CTT GGT (SEQ IDNO:223).

[0193] The assembled V_(L) and V_(H) regions were introduced into a Fabexpression vector by mutagenesis. Briefly, the non-biotinylated minusstrands were isolated after binding the PCR products toNeutrAvidin-conjugated magnetic beads and introduced into the Fabexpression vector IX-104CSA by hybridization mutagenesis (Kristensson etal., Vaccines 95, pp. 39-43, Cold Spring Harbor Laboratory, Cold SpringHarbor (1995); Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Wuet al., J. Mol. Bio. 294:151-162 (1999)).

[0194] Three humanization-CDR3-mutation libraries were constructed foreach the HUI77 and HUIV26 antibodies. The three libraries introducedrandom mutations but differed in CDR3 mutations. One library hadmutations only in LCDR3, the second library had mutations only in HCDR3,and the third library had mutations in both LCDR3 and HCDR3.

[0195] Methods essentially the same as those described above for CDR3mutagenesis were also performed on CDR1 and CDR2 of the HUIV26 and HUI77antibodies. After assembling into a Fab expression vector, the Fabscontaining HUIV26 and HUI77 variant CDRs were expressed in bacteria andtested for binding to denatured collagen. The mutant libraries werescreened with filter lift screening and ELISA. The assays were performedessentially as described previously (Huse et al., J. Immunol.149:3914-3920 (1992); Watkins et al., Anal. Biochem. 253:37-45 (1997)).Briefly, nitrocellulose membranes were pre-coated with heat-denaturedhuman collagen I or IV and used to lift E. coli-expressed variant FABsfrom phage plates. The membranes were then incubated with antibodies,either anti-human kappa chain or anti-hemaglutinin (HA) tag conjugatedto alkaline phosphatase to detect bound variant Fabs. Positive cloneswere screened again by single point ELISA (Watkins et al., supra, 1997)for binding to denatured-biotinylated human collagen I and IV,correspondingly. Beneficial variants were characterized for binding toboth collagens in native and heat-denatured forms by ELISA. Beneficialmutations were determined as those having higher affinity binding todenatured collagen relative to the corresponding wild type Fab, asdemonstrated by ELISA.

[0196] Shown in FIGS. 4B and 5B is a summary of beneficial CDR mutationsin the HUIV26 and HUI77 antibodies, respectively. FIG. 4B summarizesbeneficial single amino acid mutations in heavy chain CDR1, CDR2, andCDR3 and light chain CDR1 and CDR3 of HUIV26. An exemplary HUIV26variant having a single amino acid substitution is the 12F10Q variant,which exhibited k_(on) of 0.055 and k_(off) of 0.049 as estimated by thefold improvement based on shifts in half-maximal binding obtained fromELISA titrations.

[0197]FIG. 5B summarizes beneficial single amino acid mutations in heavychain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of HUI77.As can be seen, numerous single amino acid mutations in various CDRswere found to maintain or enhance binding to a cryptic collagen site.

[0198] This example describes CDR variants of HUIV26 and HUI77 havingbeneficial mutations.

EXAMPLE III Identification of Combinatorial Variants of HUIV26 and HUI77Antibodes having Enhanced Activity

[0199] This example describes the generation and identification ofcombinatorial variants incorporating various beneficial CDR mutations inHUIV26 and HUI77.

[0200] To further optimize HUIV26 and HUI77 antibody CDR variants,combinatorial variants, which incorporate at least two CDRs containingone or more mutations, were generated and tested for binding to acryptic collagen site. Combinatorial variants were synthesized usingprimers with one or more positions encoding variant amino acids asdescribed in Example II. The primers used are shown in FIGS. 6 and 7.

[0201] Shown in FIGS. 6 and 7 is a summary of the beneficialcombinatorial variants of HUIV26 and HUI77 antibodies, respectively. Thek_(on) and k_(off) values shown in FIGS. 6 and 7 (“SPEKon” and“SPEkoff”) were estimated as the fold improvement of variants based onshifts in half-maximal binding obtained from ELISA titrations. Alsoshown are several variants having the same beneficial CDR mutations buthaving different framework sequences. These results show that beneficialCDR mutations can be grafted into a variety of frameworks and can retainor have improved binding activity.

[0202] This example shows the generation of combinatorial CDR variantsof HUIV26 and HUI77. A number of variants were identified havingincreased affinity relative to wild type forms of the respectiveantibodies.

EXAMPLE IV Binding Activity and Specificity of HUIV26 and HUI77 Variants

[0203] This example describes the binding activity and specificity ofHUIV26 and HUI77 antibodies on native and denatured collagen.

[0204] The activity and specificity of wild type and selected exemplaryHUIV26 and HUI77 variants were determined. As shown in FIG. 8, theactivity and specificity of IX-IV26, a Fab containing wild type HUIV26CDRs, and the HUIV26 variants 2D4H1-C3 and DhuG5 were determined. Theantibodies were tested for binding to denatured collagen IV (FIG. 8A),denatured collagen I (FIG. 8B), and native collagen IV (FIG. 8C). Noneof the antibodies had significant binding activity for native collagenIV (FIG. 8C). All three antbodies exhibited binding activity fordenatured collagen IV (FIG. 8A). However, the 2D4H1-C3 and DhuG5variants exhibited significantly increased binding activity relative toIX-IV26 (FIG. 8A). IX-IV26 did not exhibit significant binding activityto denatured collagen I, and 2D4H1-C3 and DhuG5 exhibited low bindingactivity at the highest measured concentration of antibody (FIG. 8B).These results indicate that the HUIV26 variants have similar bindingactivity and specificity as that of wild type HUIV26 and maintainactivity and specificity for a cryptic collagen epitope. These resultsfurther show that variants having mutated CDRs can have maintained orincreased binding affinity relative to wild type.

[0205] As shown in FIG. 9, the activity and specificity of IX-177, a Fabcontaining wild type HUI77 CDRs, and the HUI77 variants Qh2b-B7 andQhuD9 were determined. The antibodies were tested for binding todenatured collagen I (FIG. 9A) denatured collagen IV (FIG. 9B) andnative collagen I (FIG. 9C), and the results indicate that thesevariants exhibited similar binding specificities as wild type. NeitherIX-177 nor Qhu2b-B7 exhibited significant binding activity for nativecollagen I, although the variant QhuD9 exhibited modest binding activityto native collagen at higher concentrations of antibody. The antibodiesall exhibited binding activity for denatured collagen I (FIG. 9A) anddenatured collagen IV (FIG. 9B). However, the Qhu2b-B7 and QhuD9variants exhibited significantly increased binding activity relative toIX-177 on both denatured collagen I and IV. These results indicate thatvariants having mutated CDRs can have maintained or increased bindingaffinity relative to wild type.

[0206] To further examine the effect of CDR mutations on bindingactivity, the HUIV26 variant DhuH8 was selected and expressed in twoforms, as a Fab and immunoglobulin (IgG). The binding activity of thesetwo forms was determined for native (n-IV) and denatured (d-IV) humancollagen IV. As shown in FIG. 10, neither the Fab nor IgG form of theDhu8 variant exhibited significant binding to native collagen IV. TheFab form exhibited binding activity for denatured collagen IV, and thebinding affinity was significantly increased for the IgG form. Theseresults indicate that a HUIV26 variant having one or more CDR amino acidsubstitutions relative to wild type can exhibit binding to a crypticcollagen epitope and that the binding affinity can be significantlyincreased in the IgG form relative to the Fab form of the antibodyvariant.

[0207] These results indicate that HUIV26 and HUI77 variants having oneor more CDR amino acid substitutions can exhibit similar bindingspecificity and increased binding affinity relative to wild type.

EXAMPLE V Generation of Grafted HUIV26 and HUI77 Antibodies havingOptimized CDRs

[0208] This example describes the generation of humanized HUIV26 andHUI77 antibodies incorporating beneficial CDR mutations.

[0209] A CDR variant have a beneficial mutation is identified asdescribed in Examples II and III. Once a beneficial CDR variant isidentified, the CDR variant is grafted into a human framework sequence.In addition to the CDR variant having a beneficial mutation, other CDRscan be a wild type sequence of the respective antibody or one or morevariant CDRs. At least one of the CDRs will be a variant containing abeneficial mutation. For example, if the grafted antibody contains aheavy and light chain, at least one of the heavy or light chain CDRswill have at least one amino acid mutation relative to the correspondingwild type CDR.

[0210] A human framework sequence is selected as the recipient forgrafting. The human framework can be closely related to the donorantibody framework sequence or can be relatively divergent from theparental donor antibody. Once a human framework is selected forgrafting, overlapping oligonucleotides are synthesized encoding theselected human framework and the appropriate donor CDRs, including atleast one variant CDR containing at least one beneficial mutation. Theoverlapping oligonucleotides are used to assemble a nucleic acidencoding a variable region including the selected human framework, theCDR variant, and appropriate other CDRs to generate an antibody orfragment having binding activity for a cryptic collagen site.

[0211] The assembled variable region is cloned into an expressionvector, for example, a Fab expression vector such as described inExample II, and binding activity to denatured collagen is tested, asdescribed in Examples II and III.

[0212] This example describes the generation of humanized antibodiescontaining beneficial CDR mutations of HUIV26 and HUI77 antibodies.

EXAMPLE VI Inhibition of B16 Melanoma Cell Proliferation by a VariantHUI77 Antibody

[0213] This example describes the effect of the HUI77 variant QH2b onB16 melanoma cell proliferation.

[0214] The humanized Fab designated QH2b, which is the QH2b-B7 variantof the HUI77 antibody, was engineered into a full length IgG1 antibody(QH2b-IgG1). The QH2b-IgG1 antibody was expressed in mammalian cellculture in NSO cells and purified.

[0215] The purified QH2b-IgG1 antibody was used in a cell proliferationassay in vitro. B16 melanoma cells were plated on denatured human Type Icollagen. QH2b-IgG1 (100 μg/ml/day) was added to one set of culturedishes and cell numbers were determined at the indicated times (FIG.11). As a control, the cells were not treated with antibody.

[0216] As shown in FIG. 11, B16 melanoma cells proliferated on denaturedcollagen type-I, as indicated by the increase in cell numbers over 3days. The B16 melanoma cell cultures treated with QH2b-IgG1 exhibitedessentially no cell growth over a period of 3 days, indicating that themelanoma cells did not proliferate in the presence of the HUI77 variantQH2b-IgG1.

[0217] These results indicate that a HUI77 variant having one or moreCDR amino acid substitutions can inhibit cell proliferation of B16melanoma cells.

[0218] Throughout this application various publications have beenreferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference in this application in order tomore fully describe the state of the art to which this inventionpertains. Although the invention has been described with reference tothe examples provided above, it should be understood that variousmodifications can be made without departing from the spirit of theinvention.

1 358 1 339 DNA Mus musculus CDS (1)...(339) 1 gac att gtg atg aca cagtct cca tct ttg ttg agt gtg tca gca gga 48 Asp Ile Val Met Thr Gln SerPro Ser Leu Leu Ser Val Ser Ala Gly 1 5 10 15 gag aag gtc act atg agctgc aag tcc agt cag agt ctg tta aac agt 96 Glu Lys Val Thr Met Ser CysLys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 gga aat caa aag aac tac ttggcc tgg tac cag cag aaa cca ggg cag 144 Gly Asn Gln Lys Asn Tyr Leu AlaTrp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 cct cct aaa ctg ttg atc tat ggggca tcc act agg gaa tct ggg gtc 192 Pro Pro Lys Leu Leu Ile Tyr Gly AlaSer Thr Arg Glu Ser Gly Val 50 55 60 cct gat cgc ttc aca ggc agt gga tctgga acc gat ttc act ctt atc 240 Pro Asp Arg Phe Thr Gly Ser Gly Ser GlyThr Asp Phe Thr Leu Ile 65 70 75 80 atc agc agt gtg cag gct gaa gac ctggca gtt tat tac tgt cag aat 288 Ile Ser Ser Val Gln Ala Glu Asp Leu AlaVal Tyr Tyr Cys Gln Asn 85 90 95 gat cat agt tat ccg tac acg ttc gga gggggg acc aag ctg gaa ata 336 Asp His Ser Tyr Pro Tyr Thr Phe Gly Gly GlyThr Lys Leu Glu Ile 100 105 110 aaa 339 Lys 2 113 PRT Mus musculus 2 AspIle Val Met Thr Gln Ser Pro Ser Leu Leu Ser Val Ser Ala Gly 1 5 10 15Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ile 65 70 7580 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 9095 Asp His Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100105 110 Lys 3 360 DNA Mus musculus CDS (1)...(360) 3 gag gtg aag ctt ctcgag tct gga ggt ggc ctg gtg cag cct gga gga 48 Glu Val Lys Leu Leu GluSer Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 tcc ctg aaa ctc tcctgt gca gcc tca gga ttc gat ttt agt aga tac 96 Ser Leu Lys Leu Ser CysAla Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30 tgg atg agt tgg gtc cggcag gct cca ggg aaa ggg cta gaa tgg att 144 Trp Met Ser Trp Val Arg GlnAla Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 gga gaa att aat cca gat agcagt acg ata aac tat acg cca tct cta 192 Gly Glu Ile Asn Pro Asp Ser SerThr Ile Asn Tyr Thr Pro Ser Leu 50 55 60 aag gat aaa ttc atc atc tcc agagac aac gcc aaa aat acg ctg tac 240 Lys Asp Lys Phe Ile Ile Ser Arg AspAsn Ala Lys Asn Thr Leu Tyr 65 70 75 80 ctg caa atg agc aaa gtg aga tctgag gac aca gcc ctt tat tac tgt 288 Leu Gln Met Ser Lys Val Arg Ser GluAsp Thr Ala Leu Tyr Tyr Cys 85 90 95 gca aga ccg gtt gat ggt tac tac gatgct atg gac tac tgg ggt caa 336 Ala Arg Pro Val Asp Gly Tyr Tyr Asp AlaMet Asp Tyr Trp Gly Gln 100 105 110 gga acc tca gtc acc gtc tcc tca 360Gly Thr Ser Val Thr Val Ser Ser 115 120 4 120 PRT Mus musculus 4 Glu ValLys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 SerLeu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30 TrpMet Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 GlyGlu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu 50 55 60 LysAsp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Ala Arg Pro Val Asp Gly Tyr Tyr Asp Ala Met Asp Tyr Trp Gly Gln 100 105110 Gly Thr Ser Val Thr Val Ser Ser 115 120 5 305 DNA Homo sapiens 5 gacatc gtg atg acc cag tct cca gac tcc ctg gct gtg tct ctg ggc 48 gag agggcc acc atc aac tgc aag tcc agc cag agt gtt tta tac agc 96 tcc aac aataag aac tac tta gct tgg tac cag cag aaa cca gga cag 144 cct cct aag ctgctc att tac tgg gca tct acc cgg gaa tcc ggg gtc 192 cct gac cga ttc agtggc agc ggg tct ggg aca gat ttc act ctc acc 240 atc agc agc ctg cag gctgaa gat gtg gca gtt tat tac tgt cag caa 288 tat tat agt act cct cc 305 6113 PRT Homo sapiens 6 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu AlaVal Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser GlnSer Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr GlnGln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser ThrArg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly ThrAsp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val AlaVal Tyr Tyr Cys Gln Gln 85 90 95 Asp His Ser Tyr Pro Tyr Thr Phe Gly GlnGly Thr Lys Leu Glu Ile 100 105 110 Lys 7 294 DNA Homo sapiens 7 gag gtgcag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggg ggg 48 tcc ctg agactc tcc tgt gca gcc tct gga ttc acc ttt agt agc tat 96 tgg atg agc tgggtc cgc cag gct cca ggg aag ggg ctg gag tgg gtg 144 gcc aac ata aag caagat gga agt gag aaa tac tat gtg gac tct gtg 192 aag ggc cga ttc acc atctcc aga gac aac gcc aag aac tca ctg tat 240 ctg caa atg aac agc ctg agagcc gag gac acg gct gtg tat tac tgt 288 gcg aga 294 8 120 PRT Homosapiens 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe SerSer Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Val 35 40 45 Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val AspSer Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn SerLeu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala ValTyr Tyr Cys 85 90 95 Ala Arg Pro Asp Tyr Tyr Tyr Tyr Tyr Gly Met Asp ValTrp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 9 336DNA Mus musculus CDS (1)...(336) 9 gat gtt ttg atg acc caa act cca ctctcc ctg cct gtc agt ctt gga 48 Asp Val Leu Met Thr Gln Thr Pro Leu SerLeu Pro Val Ser Leu Gly 1 5 10 15 gat caa gcc tcc atc tct tgc aga tctagt cag agc att gta cat agt 96 Asp Gln Ala Ser Ile Ser Cys Arg Ser SerGln Ser Ile Val His Ser 20 25 30 aat gga aac acc tat tta gaa tgg tac ctgcag aaa cca ggc cag tct 144 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu GlnLys Pro Gly Gln Ser 35 40 45 cca aag ctc ctg atc tac aaa gtt tcc aac cgattt tct ggt gtc cca 192 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg PheSer Gly Val Pro 50 55 60 gac agg ttc agt ggc agt gga tca ggg aca gat ttcaca ctc aag atc 240 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe ThrLeu Lys Ile 65 70 75 80 agc aga gtg gag gct gag gat ctg gga gtt tat tactgc ttt caa ggt 288 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr CysPhe Gln Gly 85 90 95 tca cat gtt ccg tgg acg ttc ggt gga ggc acc aag ctggaa atc aaa 336 Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu GluIle Lys 100 105 110 10 112 PRT Mus musculus 10 Asp Val Leu Met Thr GlnThr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser IleSer Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr TyrLeu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu IleTyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser GlySer Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val GluAla Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val ProTrp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 11 369 DNAMus musculus CDS (1)...(369) 11 cag gtt act ctg aaa gag act ggc cct gggata ttg cag ccc tcc cag 48 Gln Val Thr Leu Lys Glu Thr Gly Pro Gly IleLeu Gln Pro Ser Gln 1 5 10 15 acc ctc agt ctg act tgt tct ttc tct gggttt tca ctg agc act tct 96 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly PheSer Leu Ser Thr Ser 20 25 30 ggt atg ggt gta ggc tgg att cgt cag cct tcagga gag ggt cta gag 144 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser GlyGlu Gly Leu Glu 35 40 45 tgg ctg gca gac att tgg tgg gat gac aat aag tactat aac cca tcc 192 Trp Leu Ala Asp Ile Trp Trp Asp Asp Asn Lys Tyr TyrAsn Pro Ser 50 55 60 ctg aag agc cgg ctc aca atc tcc aag gat acc tcc agcaac cag gta 240 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser AsnGln Val 65 70 75 80 ttc ctc aag atc acc agt gtg gac act gca gat act gccact tac tac 288 Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala ThrTyr Tyr 85 90 95 tgt gct cga aga gct aac tat ggt aac ccc tac tat gct atggac tac 336 Cys Ala Arg Arg Ala Asn Tyr Gly Asn Pro Tyr Tyr Ala Met AspTyr 100 105 110 tgg ggt caa gga acc tca gtc acc gtc tcc tca 369 Trp GlyGln Gly Thr Ser Val Thr Val Ser Ser 115 120 12 123 PRT Mus musculus 12Gln Val Thr Leu Lys Glu Thr Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 1015 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 2530 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Glu Gly Leu Glu 35 4045 Trp Leu Ala Asp Ile Trp Trp Asp Asp Asn Lys Tyr Tyr Asn Pro Ser 50 5560 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val 65 7075 80 Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 8590 95 Cys Ala Arg Arg Ala Asn Tyr Gly Asn Pro Tyr Tyr Ala Met Asp Tyr100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 13 305DNA Homo sapiens 13 gat att gtg atg acc cag act cca ctc tcc ctg ccc gtcacc cct gga 48 gag ccg gcc tcc atc tcc tgc agg tct agt cag agc ctc ttggat agt 96 gat gat gga aac acc tat ttg gac tgg tac ctg cag aag cca gggcag 144 tct cca cag ctc ctg atc tat acg ctt tcc tat cgg gcc tct gga gtc192 cca gac agg ttc agt ggc agt ggg tca ggc act gat ttc aca ctg aaa 240atc agc agg gtg gag gct gag gat gtt gga gtt tat tac tgc atg caa 288 cgtata gag ttt cct tc 305 14 111 PRT Homo sapiens 14 Asp Ile Val Met ThrGln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala SerIle Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Asn ThrTyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu LeuIle Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg ValGlu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser 85 90 95 His Val ProTrp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 15 288 DNAHomo sapiens 15 caggtcacct tgaaggagtc tggtcctgcg ctggtgaaac ccacacagaccctcacactg 60 acctgcacct tctctgggtt ctcactcagc actagtggaa tgcgtgtgagctggatccgt 120 cagcccccag ggaaggccct ggagtggctt gcacgcattg attggg atgatg ata 175 aat tct aca gca cat ctc tga agaccaggct caccatctcc aaggacacct226 ccaaaaacca ggtggtcctt acaatgacca acatggaccc tgtggacaca gccacgtatt286 ac 288 16 123 PRT Homo sapiens 16 Gln Val Thr Leu Lys Glu Ser GlyPro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys ThrPhe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Arg Val Ser Trp IleArg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Arg Ile Asp TrpAsp Asp Asp Lys Phe Tyr Ser Thr Ser 50 55 60 Leu Lys Thr Arg Leu Thr IleSer Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr AsnMet Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Arg Ala AsnTyr Tyr Tyr Tyr Tyr Tyr Ala Met Asp Val 100 105 110 Trp Gly Gln Gly ThrThr Val Thr Val Ser Ser 115 120 17 340 DNA Homo sapiens CDS (1)...(339)17 gat att gtg atg acc cag act cca ctc tcc ctg ccc gtc acc cct gga 48Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 1015 gag ccg gcc tcc atc tcc tgc agg tct agt cag agc ctc ttg gat agt 96Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30gat gat gga aac acc tat ttg gac tgg tac ctg cag aag cca ggg cag 144 AspAsp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 tctcca cag ctc ctg atc tat acg ctt tcc tat cgg gcc tct gga gtc 192 Ser ProGln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val 50 55 60 cca gacagg ttc agt ggc agt ggg tca ggc act gat ttc aca ctg aaa 240 Pro Asp ArgPhe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 atc agcagg gtg gag gct gag gat gtt gga gtt tat tac tgc atg caa 288 Ile Ser ArgVal Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln 85 90 95 cgg ttc acatgt tcc gtg gac gtt cgg cca agg gac caa ggt gga aat 336 Arg Phe Thr CysSer Val Asp Val Arg Pro Arg Asp Gln Gly Gly Asn 100 105 110 caa a 340Gln 18 113 PRT Homo sapiens 18 Asp Ile Val Met Thr Gln Thr Pro Leu SerLeu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg SerSer Gln Ser Leu Leu Asp Ser 20 25 30 Asp Asp Gly Asn Thr Tyr Leu Asp TrpTyr Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Thr LeuSer Tyr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly SerGly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu Ala Glu AspVal Gly Val Tyr Tyr Cys Met Gln 85 90 95 Arg Phe Thr Cys Ser Val Asp ValArg Pro Arg Asp Gln Gly Gly Asn 100 105 110 Gln 19 51 DNA Mus musculusCDS (1)...(51) 19 aag tcc agt cag agt ctg tta aac agt gga aat caa aagaac tac ttg 48 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys AsnTyr Leu 1 5 10 15 gcc 51 Ala 20 17 PRT Mus musculus 20 Lys Ser Ser GlnSer Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 21 21 DNAMus musculus CDS (1)...(21) 21 ggg gca tcc act agg gaa tct 21 Gly AlaSer Thr Arg Glu Ser 1 5 22 7 PRT Mus musculus 22 Gly Ala Ser Thr Arg GluSer 1 5 23 27 DNA Mus musculus CDS (1)...(27) 23 cag aat gat cat agt tatccg tac acg 27 Gln Asn Asp His Ser Tyr Pro Tyr Thr 1 5 24 9 PRT Musmusculus 24 Gln Asn Asp His Ser Tyr Pro Tyr Thr 1 5 25 30 DNA Musmusculus CDS (1)...(30) 25 gga ttc gat ttt agt aga tac tgg atg agt 30Gly Phe Asp Phe Ser Arg Tyr Trp Met Ser 1 5 10 26 10 PRT Mus musculus 26Gly Phe Asp Phe Ser Arg Tyr Trp Met Ser 1 5 10 27 51 DNA Mus musculusCDS (1)...(51) 27 gaa att aat cca gat agc agt acg ata aac tat acg ccatct cta aag 48 Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro SerLeu Lys 1 5 10 15 gat 51 Asp 28 17 PRT Mus musculus 28 Glu Ile Asn ProAsp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu Lys 1 5 10 15 Asp 29 33 DNAMus musculus CDS (1)...(33) 29 ccg gtt gat ggt tac tac gat gct atg gactac 33 Pro Val Asp Gly Tyr Tyr Asp Ala Met Asp Tyr 1 5 10 30 11 PRT Musmusculus 30 Pro Val Asp Gly Tyr Tyr Asp Ala Met Asp Tyr 1 5 10 31 48 DNAMus musculus CDS (1)...(48) 31 aga tct agt cag agc att gta cat agt aatgga aac acc tat tta gaa 48 Arg Ser Ser Gln Ser Ile Val His Ser Asn GlyAsn Thr Tyr Leu Glu 1 5 10 15 32 16 PRT Mus musculus 32 Arg Ser Ser GlnSer Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 33 21 DNA Musmusculus CDS (1)...(21) 33 aaa gtt tcc aac cga ttt tct 21 Lys Val SerAsn Arg Phe Ser 1 5 34 7 PRT Mus musculus 34 Lys Val Ser Asn Arg Phe Ser1 5 35 27 DNA Mus musculus CDS (1)...(27) 35 ttt caa ggt tca cat gtt ccgtgg acg 27 Phe Gln Gly Ser His Val Pro Trp Thr 1 5 36 9 PRT Mus musculus36 Phe Gln Gly Ser His Val Pro Trp Thr 1 5 37 36 DNA Mus musculus CDS(1)...(36) 37 ggg ttt tca ctg agc act tct ggt atg ggt gta ggc 36 Gly PheSer Leu Ser Thr Ser Gly Met Gly Val Gly 1 5 10 38 12 PRT Mus musculus 38Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly 1 5 10 39 48 DNA Musmusculus CDS (1)...(48) 39 gac att tgg tgg gat gac aat aag tac tat aaccca tcc ctg aag agc 48 Asp Ile Trp Trp Asp Asp Asn Lys Tyr Tyr Asn ProSer Leu Lys Ser 1 5 10 15 40 16 PRT Mus musculus 40 Asp Ile Trp Trp AspAsp Asn Lys Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 41 39 DNA Musmusculus CDS (1)...(39) 41 aga gct aac tat ggt aac ccc tac tat gct atggac tac 39 Arg Ala Asn Tyr Gly Asn Pro Tyr Tyr Ala Met Asp Tyr 1 5 10 4213 PRT Mus musculus 42 Arg Ala Asn Tyr Gly Asn Pro Tyr Tyr Ala Met AspTyr 1 5 10 43 10 PRT Artificial Sequence synthetic antibody mutation 43Gly Phe Asp Phe Ser His Tyr Trp Met Ser 1 5 10 44 10 PRT ArtificialSequence synthetic antibody mutation 44 Gly Phe Asp Phe Ser Arg Tyr TrpIle Ser 1 5 10 45 10 PRT Artificial Sequence synthetic antibody mutation45 Gly Phe Asp Phe Ser Arg Tyr Trp Met Thr 1 5 10 46 10 PRT ArtificialSequence synthetic antibody mutation 46 Gly Phe Asp Phe Ser Arg Tyr TrpMet Ala 1 5 10 47 10 PRT Artificial Sequence Artificial sequence 47 GlyPhe Asp Phe Ser Arg Tyr Trp Met Gly 1 5 10 48 17 PRT Artificial SequenceArtificial sequence 48 Glu Ile Asn Pro Asp Ser Ser Thr Ala Asn Tyr ThrPro Ser Leu Lys 1 5 10 15 Asp 49 17 PRT Artificial Sequence Artificialsequence 49 Glu Ile Asn Pro Asp Ser Ser Thr Ser Asn Tyr Thr Pro Ser LeuAsp 1 5 10 15 Lys 50 17 PRT Artificial Sequence Artificial sequence 50Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Tyr Leu Lys 1 5 1015 Asp 51 17 PRT Artificial Sequence synthetic antibody mutation 51 GluIle Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ala Leu Lys 1 5 10 15Asp 52 17 PRT Artificial Sequence Artificial sequence 52 Glu Ile Asn ProAsp Ser Ser Thr Ile Asn Tyr Thr Pro His Leu Lys 1 5 10 15 Asp 53 17 PRTArtificial Sequence synthetic antibody mutation 53 Glu Ile Asn Pro AspSer Ser Thr Ile Asn Tyr Thr Pro Gly Leu Lys 1 5 10 15 Asp 54 17 PRTArtificial Sequence synthetic antibody mutation 54 Glu Ile Asn Pro AspSer Ser Thr Ile Asn Tyr Thr Pro Ser Leu Gln 1 5 10 15 Asp 55 17 PRTArtificial Sequence synthetic antibody mutation 55 Glu Ile Asn Pro AspSer Ser Thr Ile Asn Tyr Thr Pro Ser Leu Lys 1 5 10 15 Ser 56 11 PRTArtificial Sequence synthetic antibody mutation 56 Pro Val Pro Gly TyrTyr Asp Ala Met Asp Tyr 1 5 10 57 11 PRT Artificial Sequence syntheticantibody mutation 57 Pro Val Gly Gly Tyr Tyr Asp Ala Met Asp Tyr 1 5 1058 11 PRT Artificial Sequence synthetic antibody mutation 58 Pro Val ThrGly Tyr Tyr Asp Ala Met Asp Tyr 1 5 10 59 11 PRT Artificial Sequencesynthetic antibody mutation 59 Pro Val Ala Gly Tyr Tyr Asp Ala Met AspTyr 1 5 10 60 11 PRT Artificial Sequence synthetic antibody mutation 60Pro Val Asp Pro Tyr Tyr Asp Ala Met Asp Tyr 1 5 10 61 11 PRT ArtificialSequence synthetic antibody mutation 61 Pro Val Asp Ala Tyr Tyr Asp AlaMet Asp Tyr 1 5 10 62 11 PRT Artificial Sequence synthetic antibodymutation 62 Pro Val Asp His Tyr Tyr Asp Ala Met Asp Tyr 1 5 10 63 11 PRTArtificial Sequence synthetic antibody mutation 63 Pro Val Asp Gly TyrTyr Asp Ala Met Asp Pro 1 5 10 64 11 PRT Artificial Sequence Artificialsequence 64 Pro Val Asp Gly Tyr Tyr Asp Ala Met Asp Asn 1 5 10 65 17 PRTArtificial Sequence synthetic antibody mutation 65 Lys Ser Ser Arg SerLeu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 66 17 PRTArtificial Sequence synthetic antibody mutation 66 Lys Ser Ser Ser SerLeu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 67 17 PRTArtificial Sequence synthetic antibody mutation 67 Lys Ser Ser Gln SerLeu Leu Ser Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 68 17 PRTArtificial Sequence synthetic antibody mutation 68 Lys Ser Ser Gln SerLeu Leu Asn Tyr Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 69 17 PRTArtificial Sequence synthetic antibody mutation 69 Lys Ser Ser Gln SerLeu Leu Asn Trp Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 70 17 PRTArtificial Sequence synthetic antibody mutation 70 Lys Ser Ser Gln SerLeu Leu Asn His Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 71 17 PRTArtificial Sequence synthetic antibody mutation 71 Lys Ser Ser Gln SerLeu Leu Asn Arg Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 72 17 PRTArtificial Sequence synthetic antibody mutation 72 Lys Ser Ser Gln SerLeu Leu Asn Ser Tyr Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 73 17 PRTArtificial Sequence synthetic antibody mutation 73 Lys Ser Ser Gln SerLeu Leu Asn Ser Arg Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 74 17 PRTArtificial Sequence synthetic antibody mutation 74 Lys Ser Ser Gln SerLeu Leu Asn Ser His Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 75 17 PRTArtificial Sequence synthetic antibody mutation 75 Lys Ser Ser Gln SerLeu Leu Asn Ser Ile Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 76 17 PRTArtificial Sequence synthetic antibody mutation 76 Lys Ser Ser Gln SerLeu Leu Asn Ser Gly Asn Lys Lys Asn Tyr Leu 1 5 10 15 Ala 77 9 PRTArtificial Sequence synthetic antibody mutation 77 Gln Asn Asp His GlnTyr Pro Tyr Thr 1 5 78 9 PRT Artificial Sequence synthetic antibodymutation 78 Gln Asn Asp His Gly Tyr Pro Tyr Thr 1 5 79 9 PRT ArtificialSequence synthetic antibody mutation 79 Gln Asn Asp His Leu Tyr Pro TyrThr 1 5 80 9 PRT Artificial Sequence synthetic antibody mutation 80 GlnAsn Asp His Ala Tyr Pro Tyr Thr 1 5 81 9 PRT Artificial Sequencesynthetic antibody mutation 81 Gln Asn Asp His Thr Tyr Pro Tyr Thr 1 582 9 PRT Artificial Sequence synthetic antibody mutation 82 Gln Asn AspHis Val Tyr Pro Tyr Thr 1 5 83 9 PRT Artificial Sequence syntheticantibody mutation 83 Gln Asn Asp His Ser Asn Pro Tyr Thr 1 5 84 9 PRTArtificial Sequence synthetic antibody mutation 84 Gln Asn Asp His SerSer Pro Tyr Thr 1 5 85 9 PRT Artificial Sequence synthetic antibodymutation 85 Gln Asn Asp His Ser Pro Pro Tyr Thr 1 5 86 9 PRT ArtificialSequence synthetic antibody mutation 86 Gln Asn Asp His Ser Met Pro TyrThr 1 5 87 12 PRT Artificial Sequence synthetic antibody mutation 87 GlyPhe Ser Leu Ser Thr Pro Gly Met Gly Val Gly 1 5 10 88 12 PRT ArtificialSequence synthetic antibody mutation 88 Gly Phe Ser Leu Ser Thr Trp GlyMet Gly Val Gly 1 5 10 89 12 PRT Artificial Sequence synthetic antibodymutation 89 Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Trp 1 5 10 90 12PRT Artificial Sequence synthetic antibody mutation 90 Gly Phe Ser LeuSer Thr Ser Gly Met Gly Val Leu 1 5 10 91 12 PRT Artificial Sequencesynthetic antibody mutation 91 Gly Phe Ser Leu Ser Thr Ser Gly Met GlyVal Ala 1 5 10 92 16 PRT Artificial Sequence synthetic antibody mutation92 Asp Ile Trp Trp Asp Asp Asn Lys Tyr Ser Asn Pro Ser Leu Lys Ser 1 510 15 93 16 PRT Artificial Sequence synthetic antibody mutation 93 AspIle Trp Trp Asp Asp Asn Lys Tyr Ala Asn Pro Ser Leu Lys Ser 1 5 10 15 9416 PRT Artificial Sequence synthetic antibody mutation 94 Asp Ile TrpTrp Asp Asp Asn Lys Tyr Pro Asn Pro Ser Leu Lys Ser 1 5 10 15 95 16 PRTArtificial Sequence synthetic antibody mutation 95 Asp Ile Trp Trp AspAsp Asn Lys Tyr Tyr Asn Pro Ser Leu Pro Ser 1 5 10 15 96 13 PRTArtificial Sequence synthetic antibody mutation 96 Pro Ala Asn Tyr GlyAsn Pro Tyr Tyr Ala Met Asp Tyr 1 5 10 97 13 PRT Artificial Sequencesynthetic antibody mutation 97 Gln Ala Asn Tyr Gly Asn Pro Tyr Tyr AlaMet Asp Tyr 1 5 10 98 13 PRT Artificial Sequence synthetic antibodymutation 98 Leu Ala Asn Tyr Gly Asn Pro Tyr Tyr Ala Met Asp Tyr 1 5 1099 13 PRT Artificial Sequence synthetic antibody mutation 99 Thr Ala AsnTyr Gly Asn Pro Tyr Tyr Ala Met Asp Tyr 1 5 10 100 13 PRT ArtificialSequence synthetic antibody mutation 100 Val Ala Asn Tyr Gly Asn Pro TyrTyr Ala Met Asp Tyr 1 5 10 101 13 PRT Artificial Sequence syntheticantibody mutation 101 Arg Ala Asn Tyr Gly Val Pro Tyr Tyr Ala Met AspTyr 1 5 10 102 13 PRT Artificial Sequence synthetic antibody mutation102 Arg Ala Asn Tyr Gly Trp Pro Tyr Tyr Ala Met Asp Tyr 1 5 10 103 13PRT Artificial Sequence synthetic antibody mutation 103 Arg Ala Asn TyrGly Asn Pro Tyr Tyr Ala Gln Asp Tyr 1 5 10 104 13 PRT ArtificialSequence synthetic antibody mutation 104 Arg Ala Asn Tyr Gly Asn Pro TyrTyr Ala Asn Asp Tyr 1 5 10 105 13 PRT Artificial Sequence syntheticantibody mutation 105 Arg Ala Asn Tyr Gly Asn Pro Tyr Tyr Ala Thr AspTyr 1 5 10 106 13 PRT Artificial Sequence synthetic antibody mutation106 Arg Ala Asn Tyr Gly Asn Pro Tyr Tyr Ala Met Asp Lys 1 5 10 107 13PRT Artificial Sequence synthetic antibody mutation 107 Arg Ala Asn TyrGly Asn Pro Tyr Tyr Ala Met Asp Thr 1 5 10 108 13 PRT ArtificialSequence synthetic antibody mutation 108 Arg Ala Asn Tyr Gly Asn Pro TyrTyr Ala Met Asp Met 1 5 10 109 13 PRT Artificial Sequence syntheticantibody mutation 109 Arg Ala Asn Tyr Gly Asn Pro Tyr Tyr Ala Met AspHis 1 5 10 110 16 PRT Artificial Sequence synthetic antibody mutation110 Arg Ser Ser Gln Ser Ile Pro His Ser Asn Gly Asn Thr Tyr Leu Glu 1 510 15 111 16 PRT Artificial Sequence synthetic antibody mutation 111 ArgSer Ser Gln Ser Ile Trp His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15112 16 PRT Artificial Sequence synthetic antibody mutation 112 Arg SerSer Gln Ser Ile Val Leu Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 113 16PRT Artificial Sequence synthetic antibody mutation 113 Arg Ser Ser GlnSer Ile Val Ser Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 114 16 PRTArtificial Sequence synthetic antibody mutation 114 Arg Ser Ser Gln SerIle Val His Trp Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 115 16 PRTArtificial Sequence synthetic antibody mutation 115 Arg Ser Ser Gln SerIle Val His Ser Tyr Gly Asn Thr Tyr Leu Glu 1 5 10 15 116 16 PRTArtificial Sequence synthetic antibody mutation 116 Arg Ser Ser Gln SerIle Val His Ser Trp Gly Asn Thr Tyr Leu Glu 1 5 10 15 117 16 PRTArtificial Sequence synthetic antibody mutation 117 Arg Ser Ser Gln SerIle Val His Ser Asn Gly Tyr Thr Tyr Leu Glu 1 5 10 15 118 16 PRTArtificial Sequence synthetic antibody mutation 118 Arg Ser Ser Gln SerIle Val His Ser Asn Gly Asn Thr Tyr Phe Glu 1 5 10 15 119 16 PRTArtificial Sequence synthetic antibody mutation 119 Arg Ser Ser Gln SerIle Val His Ser Asn Gly Asn Thr Tyr Val Glu 1 5 10 15 120 7 PRTArtificial Sequence synthetic antibody mutation 120 Ser Val Ser Asn ArgPhe Ser 1 5 121 7 PRT Artificial Sequence synthetic antibody mutation121 Lys Ala Ser Asn Arg Phe Ser 1 5 122 7 PRT Artificial Sequencesynthetic antibody mutation 122 Lys Val Ser Ser Arg Phe Ser 1 5 123 7PRT Artificial Sequence synthetic antibody mutation 123 Lys Val Ser AsnLeu Phe Ser 1 5 124 7 PRT Artificial Sequence synthetic antibodymutation 124 Lys Val Ser Asn Arg Phe Trp 1 5 125 7 PRT ArtificialSequence synthetic antibody mutation 125 Lys Val Ser Asn Arg Phe Phe 1 5126 9 PRT Artificial Sequence synthetic antibody mutation 126 Val GlnGly Ser His Val Pro Trp Thr 1 5 127 9 PRT Artificial Sequence syntheticantibody mutation 127 His Gln Gly Ser His Val Pro Trp Thr 1 5 128 9 PRTArtificial Sequence synthetic antibody mutation 128 Phe Arg Gly Ser HisVal Pro Trp Thr 1 5 129 9 PRT Artificial Sequence synthetic antibodymutation 129 Phe Trp Gly Ser His Val Pro Trp Thr 1 5 130 9 PRTArtificial Sequence synthetic antibody mutation 130 Phe Gln Ser Ser HisVal Pro Trp Thr 1 5 131 9 PRT Artificial Sequence synthetic antibodymutation 131 Phe Gln Gly Trp His Val Pro Trp Thr 1 5 132 9 PRTArtificial Sequence synthetic antibody mutation 132 Phe Gln Gly Glu HisVal Pro Trp Thr 1 5 133 9 PRT Artificial Sequence synthetic antibodymutation 133 Phe Gln Gly Ser Leu Val Pro Trp Thr 1 5 134 9 PRTArtificial Sequence synthetic antibody mutation 134 Phe Gln Gly Ser ThrVal Pro Trp Thr 1 5 135 9 PRT Artificial Sequence synthetic antibodymutation 135 Phe Gln Gly Ser Ser Val Pro Trp Thr 1 5 136 9 PRTArtificial Sequence synthetic antibody mutation 136 Phe Gln Gly Ser AlaVal Pro Trp Thr 1 5 137 9 PRT Artificial Sequence synthetic antibodymutation 137 Phe Gln Gly Ser Gln Val Pro Trp Thr 1 5 138 9 PRTArtificial Sequence synthetic antibody mutation 138 Phe Gln Gly Ser HisThr Pro Trp Thr 1 5 139 9 PRT Artificial Sequence synthetic antibodymutation 139 Phe Gln Gly Ser His Val Pro Trp Ala 1 5 140 9 PRTArtificial Sequence synthetic antibody mutation 140 Phe Gln Gly Ser HisVal Pro Trp Arg 1 5 141 9 PRT Artificial Sequence synthetic antibodymutation 141 Phe Gln Gly Ser His Val Pro Trp His 1 5 142 9 PRTArtificial Sequence synthetic antibody mutation 142 Phe Gln Gly Ser HisVal Pro Trp Lys 1 5 143 9 PRT Artificial Sequence synthetic antibodymutation 143 Phe Gln Gly Ser His Val Pro Trp Ile 1 5 144 16 PRTArtificial Sequence synthetic antibody mutation 144 Asp Ile Trp Trp AspAsp Asn Lys Tyr Thr Asn Pro Ser Leu Lys Ser 1 5 10 15 145 9 PRTArtificial Sequence synthetic antibody mutation 145 Phe Gln Gly Ser HisPhe Pro Trp Thr 1 5 146 16 PRT Artificial Sequence synthetic antibodymutation 146 Arg Ser Ser Gln Ser Ile Val His Ser Gln Gly Asn Thr Tyr LeuGlu 1 5 10 15 147 12 PRT Artificial Sequence synthetic antibody mutation147 Gly Phe Ser Leu Ser Thr Pro Gly Met Gly Val Trp 1 5 10 148 12 PRTArtificial Sequence synthetic antibody mutation 148 Gly Phe Ser Leu SerThr Pro Gly Met Gly Val Ala 1 5 10 149 15 PRT Artificial Sequencesynthetic antibody mutation 149 Arg Ser Ser Gln Ser Ile Val Ser Ser TrpGly Asn Thr Tyr Leu 1 5 10 15 150 15 PRT Artificial Sequence syntheticantibody mutation 150 Arg Ser Ser Gln Ser Ile Val Ser Ser Tyr Gly AsnThr Tyr Leu 1 5 10 15 151 15 PRT Artificial Sequence synthetic antibodymutation 151 Arg Ser Ser Gln Ser Ile Val Ser Ser Gln Gly Asn Thr Tyr Leu1 5 10 15 152 15 PRT Artificial Sequence synthetic antibody mutation 152Arg Ser Ser Gln Ser Ile Val His Ser Gln Gly Asn Thr Tyr Phe 1 5 10 15153 15 PRT Artificial Sequence synthetic antibody mutation 153 Arg SerSer Gln Ser Ile Val Ser Ser Trp Gly Asn Thr Tyr Phe 1 5 10 15 154 17 PRTArtificial Sequence synthetic antibody mutation 154 Glu Ile Asn Pro AspSer Ser Thr Ala Asn Tyr Thr Pro Ala Leu Lys 1 5 10 15 Asp 155 17 PRTArtificial Sequence synthetic antibody mutation 155 Glu Ile Asn Pro AspSer Ser Thr Ala Asn Tyr Thr Pro Tyr Leu Lys 1 5 10 15 Asp 156 17 PRTArtificial Sequence synthetic antibody mutation 156 Glu Ile Asn Pro AspSer Ser Thr Ala Asn Tyr Thr Pro His Leu Lys 1 5 10 15 Asp 157 17 PRTArtificial Sequence synthetic antibody mutation 157 Lys Ser Ser Gln SerLeu Leu Asn Trp Tyr Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 158 17 PRTArtificial Sequence synthetic antibody mutation 158 Lys Ser Ser Gln SerLeu Leu Asn Tyr Tyr Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 159 17 PRTArtificial Sequence synthetic antibody mutation 159 Lys Ser Ser Gln SerLeu Leu Asn Tyr His Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 160 17 PRTArtificial Sequence synthetic antibody mutation 160 Lys Ser Ser Gln SerLeu Leu Asn Arg Tyr Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 161 17 PRTArtificial Sequence synthetic antibody mutation 161 Lys Ser Ser Gln SerLeu Leu Asn Trp His Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 162 17 PRTArtificial Sequence synthetic antibody mutation 162 Glu Ile Asn Pro AspSer Ser Thr Val Asn Tyr Thr Pro Ser Leu Lys 1 5 10 15 Asp 163 39 DNAArtificial Sequence Primer 163 tctctggaga tggtgaattt acgtactgctatctggatt 39 164 41 DNA Artificial Sequence Primer 164 ctaagtagttcttttggttg ttataacaga ctctggctgg a 41 165 51 DNA Artificial SequencePrimer 165 tggagcctgg cggacccagg hcatccaata tctactaaag gtgaatccag a 51166 65 DNA Artificial Sequence Primer 166 tctctggaga tggtgaatytatcctttagg gmtggcgtat agttggccgt actgctatct 60 ggatt 65 167 65 DNAArtificial Sequence Primer 167 tctctggaga tggtgaatyt atcctttaggtrtggcgtat agttggccgt actgctatct 60 ggatt 65 168 46 DNA ArtificialSequence Primer 168 ctaagtagtt cttttggttg trgtrgytta acagactctg gctgga46 169 46 DNA Artificial Sequence Primer 169 ctaagtagtt cttttggttgcsgtrgytta acagactctg gctgga 46 170 46 DNA Artificial Sequence Primer170 ctaagtagtt cttttggttg trgckgytta acagactctg gctgga 46 171 46 DNAArtificial Sequence Primer 171 ctaagtagtt cttttggttg csgckgyttaacagactctg gctgga 46 172 46 DNA Artificial Sequence Primer 172ctaagtagtt cttttggttg trccagytta acagactctg gctgga 46 173 46 DNAArtificial Sequence Primer 173 ctaagtagtt cttttggttg csccagyttaacagactctg gctgga 46 174 40 DNA Artificial Sequence Primer 174cttctgcagg taccattcgt tatacaatgc tctgactaga 40 175 57 DNA ArtificialSequence Primer 175 tgggggctga cggatccacm acacacccat tccacragtgctgagtgaga acccaga 57 176 57 DNA Artificial Sequence Primer 176tgggggctga cggatccags ccacacccat tccacractg ctgagtgaga acccaga 57 177 40DNA Artificial Sequence Primer 177 gctcttcaga gatgggttag vgtatttattgtcatcccac 40 178 60 DNA Artificial Sequence Primer 178 cttctgcaggtaccattcma aataggtgtt tccccaactc ratacaatgc tctgactaga 60 179 60 DNAArtificial Sequence Primer 179 cttctgcagg taccattcma aataggtgtttccgtaactc ratacaatgc tctgactaga 60 180 60 DNA Artificial SequencePrimer 180 cttctgcagg taccattcma aataggtgtt tccctgactc ratacaatgctctgactaga 60 181 60 DNA Artificial Sequence Primer 181 cttctgcaggtaccattcma aataggtgtt tccccaactg tgtacaatgc tctgactaga 60 182 60 DNAArtificial Sequence Primer 182 cttctgcagg taccattcma aataggtgtttccctaactg tctacaatgc tctgactaga 60 183 60 DNA Artificial SequencePrimer 183 cttctgcagg taccattcma aataggtgtt tccctcactg tgtacaatgctctgactaga 60 184 18 DNA Artificial Sequence Primer 184 ttggtgctgatgttctgg 18 185 18 DNA Artificial Sequence Primer 185 atcttcttgctgttctgg 18 186 18 DNA Artificial Sequence Primer 186 tgggtgctgctgctctgg 18 187 18 DNA Artificial Sequence Primer 187 gggctgcttgtgctctgg 18 188 18 DNA Artificial Sequence Primer 188 ggaatcttgttgctctgg 18 189 18 DNA Artificial Sequence Primer 189 rtrttsctgctgctrtgg 18 190 18 DNA Artificial Sequence Primer 190 ggtctcctgttgctctgt 18 191 18 DNA Artificial Sequence Primer 191 atatttctactgctctgt 18 192 18 DNA Artificial Sequence Primer 192 gtcataatrtccagagga 18 193 17 DNA Artificial Sequence Primer 193 ctgagctgtg tattcct17 194 17 DNA Artificial Sequence Primer 194 ctcarmttga ttttcct 17 19517 DNA Artificial Sequence Primer 195 tggrtcatst tcttcct 17 196 17 DNAArtificial Sequence Primer 196 tksrtctttc tcttcct 17 197 17 DNAArtificial Sequence Primer 197 tgtatcatsc tcttctt 17 198 17 DNAArtificial Sequence Primer 198 tggrtctttc tcttttt 17 199 18 DNAArtificial Sequence Primer 199 ttaaacttgg gtttttct 18 200 17 DNAArtificial Sequence Primer 200 gkgctgytcy tctgcct 17 201 18 DNAArtificial Sequence Primer 201 ttaagtcttc tgtacctg 18 202 20 DNAArtificial Sequence Primer 202 tcagtaactg caggtgtcca 20 203 20 DNAArtificial Sequence Primer 203 ttttaaaagg tgtccagtgt 20 204 20 DNAArtificial Sequence Primer 204 gcaacagcta caggtgtcca 20 205 20 DNAArtificial Sequence Primer 205 cagctacagr tgtccactcc 20 206 22 DNAArtificial Sequence Primer 206 atttccaagc tgtgtcctgt cc 22 207 23 DNAArtificial Sequence Primer 207 ctcctgtcag gaactgcagg tgt 23 208 23 DNAArtificial Sequence Primer 208 cagtggttac aggggtcaat tca 23 209 21 DNAArtificial Sequence Primer 209 ctgttsacag cchttcckgg t 21 210 21 DNAArtificial Sequence Primer 210 ctgatggcag ctgcccaaag t 21 211 20 DNAArtificial Sequence Primer 211 tttatcaagg tgtgcattgt 20 212 27 DNAArtificial Sequence Primer 212 tcactggatg gtgggaagat ggataca 27 213 24DNA Artificial Sequence Primer 213 gacatttggg aaggactgac tctc 24 214 24DNA Artificial Sequence Primer 214 cagggggctc tcgcaggaga cgag 24 215 36DNA Artificial Sequence Primer 215 atcttcttgc tgttctgggt atctggaacctgtggg 36 216 36 DNA Artificial Sequence Primer 216 ttggtgctgatgttctggat tcctgcttcc agcagt 36 217 38 DNA Artificial Sequence Primer217 gtggacgttc ggccaaggga ccaaggtgga aatcaaac 38 218 39 DNA ArtificialSequence Primer 218 tgtacacttt tggccagggg accaagctgg agatcaaac 39 219 63DNA Artificial Sequence Primer 219 attactacta ctactacggt atggacgtctggggccaagg gaccacggtc accgtctcct 60 cag 63 220 27 DNA ArtificialSequence primer 220 ttactcgctg cccaaccagc catggcc 27 221 21 DNAArtificial Sequence primer 221 gacagatggt gcagccacag t 21 222 27 DNAArtificial Sequence primer 222 ttactgttta cccctgtgac aaaagcc 27 223 21DNA Artificial Sequence primer 223 gaagaccgat gggcccttgg t 21 224 66 DNAArtificial Sequence primer 224 cttggtcccc tggccaaaag tgtacggataactatgatca ttmnnacagt aataaactgc 60 cacatc 66 225 66 DNA ArtificialSequence primer 225 cttggtcccc tggccaaaag tgtacggata actatgatcmnnctgacagt aataaactgc 60 cacatc 66 226 66 DNA Artificial Sequence primer226 cttggtcccc tggccaaaag tgtacggata actatgmnna ttctgacagt aataaactgc 60cacatc 66 227 66 DNA Artificial Sequence primer 227 cttggtcccctggccaaaag tgtacggata actmnnatca ttctgacagt aataaactgc 60 cacatc 66 22866 DNA Artificial Sequence primer 228 cttggtcccc tggccaaaag tgtacggatamnnatgatca ttctgacagt aataaactgc 60 cacatc 66 229 66 DNA ArtificialSequence primer 229 cttggtcccc tggccaaaag tgtacggmnn actatgatcattctgacagt aataaactgc 60 cacatc 66 230 66 DNA Artificial Sequence primer230 cttggtcccc tggccaaaag tgtamnnata actatgatca ttctgacagt aataaactgc 60cacatc 66 231 66 DNA Artificial Sequence primer 231 cttggtcccctggccaaaag tmnncggata actatgatca ttctgacagt aataaactgc 60 cacatc 66 23266 DNA Artificial Sequence primer 232 cttggtcccc tggccaaamn ngtacggataactatgatca ttctgacagt aataaactgc 60 cacatc 66 233 69 DNA ArtificialSequence primer 233 cgtggttcct tgcccccagt agtccatagc atcgtagtaaccatcaacmn ntctcgcaca 60 gtaatacac 69 234 69 DNA Artificial Sequenceprimer 234 cgtggttcct tgcccccagt agtccatagc atcgtagtaa ccatcmnncggtctcgcaca 60 gtaatacac 69 235 69 DNA Artificial Sequence primer 235cgtggttcct tgcccccagt agtccatagc atcgtagtaa ccmnnaaccg gtctcgcaca 60gtaatacac 69 236 69 DNA Artificial Sequence primer 236 cgtggttccttgcccccagt agtccatagc atcgtagtam nnatcaaccg gtctcgcaca 60 gtaatacac 69237 69 DNA Artificial Sequence primer 237 cgtggttcct tgcccccagtagtccatagc atcgtamnna ccatcaaccg gtctcgcaca 60 gtaatacac 69 238 69 DNAArtificial Sequence primer 238 cgtggttcct tgcccccagt agtccatagcatcmnngtaa ccatcaaccg gtctcgcaca 60 gtaatacac 69 239 69 DNA ArtificialSequence primer 239 cgtggttcct tgcccccagt agtccatagc mnngtagtaaccatcaaccg gtctcgcaca 60 gtaatacac 69 240 69 DNA Artificial Sequenceprimer 240 cgtggttcct tgcccccagt agtccatmnn atcgtagtaa ccatcaaccggtctcgcaca 60 gtaatacac 69 241 69 DNA Artificial Sequence primer 241cgtggttcct tgcccccagt agtcmnnagc atcgtagtaa ccatcaaccg gtctcgcaca 60gtaatacac 69 242 69 DNA Artificial Sequence primer 242 cgtggttccttgcccccagt amnncatagc atcgtagtaa ccatcaaccg gtctcgcaca 60 gtaatacac 69243 69 DNA Artificial Sequence primer 243 cgtggttcct tgcccccamnngtccatagc atcgtagtaa ccatcaaccg gtctcgcaca 60 gtaatacac 69 244 66 DNAArtificial Sequence primer 244 cttggtgccc tggccgaacg tccacggaacatgtgaacct tgmnngcagt aataaactcc 60 aacatc 66 245 66 DNA ArtificialSequence primer 245 cttggtgccc tggccgaacg tccacggaac atgtgaaccmnnaaagcagt aataaactcc 60 aacatc 66 246 66 DNA Artificial Sequence primer246 cttggtgccc tggccgaacg tccacggaac atgtgamnnt tgaaagcagt aataaactcc 60aacatc 66 247 66 DNA Artificial Sequence primer 247 cttggtgccctggccgaacg tccacggaac atgmnnacct tgaaagcagt aataaactcc 60 aacatc 66 24866 DNA Artificial Sequence primer 248 cttggtgccc tggccgaacg tccacggaacmnntgaacct tgaaagcagt aataaactcc 60 aacatc 66 249 66 DNA ArtificialSequence primer 249 cttggtgccc tggccgaacg tccacggmnn atgtgaaccttgaaagcagt aataaactcc 60 aacatc 66 250 66 DNA Artificial Sequence primer250 cttggtgccc tggccgaacg tccamnnaac atgtgaacct tgaaagcagt aataaactcc 60aacatc 66 251 66 DNA Artificial Sequence primer 251 cttggtgccctggccgaacg tmnncggaac atgtgaacct tgaaagcagt aataaactcc 60 aacatc 66 25266 DNA Artificial Sequence primer 252 cttggtgccc tggccgaamn nccacggaacatgtgaacct tgaaagcagt aataaactcc 60 aacatc 66 253 75 DNA ArtificialSequence primer 253 cgtggttcct tgcccccagt agtccatagc atagtaggggttaccatagt tagcmnntcg 60 agcacagtaa tacgt 75 254 75 DNA ArtificialSequence primer 254 cgtggttcct tgcccccagt agtccatagc atagtaggggttaccatagt tmnntcttcg 60 agcacagtaa tacgt 75 255 75 DNA ArtificialSequence primer 255 cgtggttcct tgcccccagt agtccatagc atagtaggggttaccatamn nagctcttcg 60 agcacagtaa tacgt 75 256 75 DNA ArtificialSequence primer 256 cgtggttcct tgcccccagt agtccatagc atagtaggggttaccmnngt tagctcttcg 60 agcacagtaa tacgt 75 257 75 DNA ArtificialSequence primer 257 cgtggttcct tgcccccagt agtccatagc atagtaggggttmnnatagt tagctcttcg 60 agcacagtaa tacgt 75 258 75 DNA ArtificialSequence primer 258 cgtggttcct tgcccccagt agtccatagc atagtagggmnnaccatagt tagctcttcg 60 agcacagtaa tacgt 75 259 75 DNA ArtificialSequence primer 259 cgtggttcct tgcccccagt agtccatagc atagtamnngttaccatagt tagctcttcg 60 agcacagtaa tacgt 75 260 75 DNA ArtificialSequence primer 260 cgtggttcct tgcccccagt agtccatagc atamnnggggttaccatagt tagctcttcg 60 agcacagtaa tacgt 75 261 75 DNA ArtificialSequence primer 261 cgtggttcct tgcccccagt agtccatagc mnngtaggggttaccatagt tagctcttcg 60 agcacagtaa tacgt 75 262 75 DNA ArtificialSequence primer 262 cgtggttcct tgcccccagt agtccatmnn atagtaggggttaccatagt tagctcttcg 60 agcacagtaa tacgt 75 263 75 DNA ArtificialSequence primer 263 cgtggttcct tgcccccagt agtcmnnagc atagtaggggttaccatagt tagctcttcg 60 agcacagtaa tacgt 75 264 75 DNA ArtificialSequence primer 264 cgtggttcct tgcccccagt amnncatagc atagtaggggttaccatagt tagctcttcg 60 agcacagtaa tacgt 75 265 75 DNA ArtificialSequence primer 265 cgtggttcct tgcccccamn ngtccatagc atagtaggggttaccatagt tagctcttcg 60 agcacagtaa tacgt 75 266 60 DNA ArtificialSequence primer 266 gttcttttgg tttccgcwgt ttaacagact ctggctggamnngcagttga tggtggccct 60 267 60 DNA Artificial Sequence primer 267gttcttttgg tttccgcwgt ttaacagact ctggctmnnc ttgcagttga tggtggccct 60 26860 DNA Artificial Sequence primer 268 gttcttttgg tttccgcwgt ttaacagactctgmnnggac ttgcagttga tggtggccct 60 269 60 DNA Artificial Sequenceprimer 269 gttcttttgg tttccgcwgt ttaacagact mnngctggac ttgcagttgatggtggccct 60 270 60 DNA Artificial Sequence primer 270 gttcttttggtttccgcwgt ttaacagmnn ctggctggac ttgcagttga tggtggccct 60 271 60 DNAArtificial Sequence primer 271 gttcttttgg tttccgcwgt ttaamnnactctggctggac ttgcagttga tggtggccct 60 272 60 DNA Artificial Sequenceprimer 272 gttcttttgg tttccgcwgt tmnncagact ctggctggac ttgcagttgatggtggccct 60 273 60 DNA Artificial Sequence primer 273 gttcttttggtttccgcwmn ntaacagact ctggctggac ttgcagttga tggtggccct 60 274 63 DNAArtificial Sequence primer 274 tggtttctgc tggtaccaag ctaagtagttcttttggttt ccmnngttta acagactctg 60 gct 63 275 63 DNA ArtificialSequence primer 275 tggtttctgc tggtaccaag ctaagtagtt cttttggttmnngcwgttta acagactctg 60 gct 63 276 63 DNA Artificial Sequence primer276 tggtttctgc tggtaccaag ctaagtagtt cttttgmnnt ccgcwgttta acagactctg 60gct 63 277 63 DNA Artificial Sequence primer 277 tggtttctgc tggtaccaagctaagtagtt cttmnngttt ccgcwgttta acagactctg 60 gct 63 278 63 DNAArtificial Sequence primer 278 tggtttctgc tggtaccaag ctaagtagttmnnttggttt ccgcwgttta acagactctg 60 gct 63 279 63 DNA ArtificialSequence primer 279 tggtttctgc tggtaccaag ctaagtamnn cttttggtttccgcwgttta acagactctg 60 gct 63 280 63 DNA Artificial Sequence primer280 tggtttctgc tggtaccaag ctaamnngtt cttttggttt ccgcwgttta acagactctg 60gct 63 281 63 DNA Artificial Sequence primer 281 tggtttctgc tggtaccaagcmnngtagtt cttttggttt ccgcwgttta acagactctg 60 gct 63 282 63 DNAArtificial Sequence primer 282 tggtttctgc tggtaccamn ntaagtagttcttttggttt ccgcwgttta acagactctg 60 gct 63 283 57 DNA ArtificialSequence primer 283 gaatcggtca gggaccccgg attccctggt agatgcmnngtaaatgagca gcttagg 57 284 57 DNA Artificial Sequence primer 284gaatcggtca gggaccccgg attccctggt agamnncccg taaatgagca gcttagg 57 285 57DNA Artificial Sequence primer 285 gaatcggtca gggaccccgg attccctggtmnntgccccg taaatgagca gcttagg 57 286 57 DNA Artificial Sequence primer286 gaatcggtca gggaccccgg attccctmnn agatgccccg taaatgagca gcttagg 57287 57 DNA Artificial Sequence primer 287 gaatcggtca gggaccccggattcmnnggt agatgccccg taaatgagca gcttagg 57 288 57 DNA ArtificialSequence primer 288 gaatcggtca gggaccccgg amnncctggt agatgccccgtaaatgagca gcttagg 57 289 57 DNA Artificial Sequence primer 289gaatcggtca gggaccccmn nttccctggt agatgccccg taaatgagca gcttagg 57 290 51DNA Artificial Sequence primer 290 tggagcctgg cggacccagc tcatccaatamnnactaaag gtgaatccag a 51 291 51 DNA Artificial Sequence primer 291tggagcctgg cggacccagc tcatccamnn tctactaaag gtgaatccag a 51 292 51 DNAArtificial Sequence primer 292 tggagcctgg cggacccagc tcatmnnatatctactaaag gtgaatccag a 51 293 51 DNA Artificial Sequence primer 293tggagcctgg cggacccagc tmnnccaata tctactaaag gtgaatccag a 51 294 51 DNAArtificial Sequence primer 294 tggagcctgg cggacccamn ncatccaatatctactaaag gtgaatccag a 51 295 67 DNA Artificial Sequence primer 295tagagatggc gtatagttta tcgtactgct atctggattt atmnngccaa yccactccag 60ccctttc 67 296 67 DNA Artificial Sequence primer 296 tagagatggcgtatagttta tcgtactgct atctggattm nnttcgccaa yccactccag 60 ccctttc 67 29767 DNA Artificial Sequence primer 297 tagagatggc gtatagttta tcgtactgctatctggmnnt atttcgccaa yccactccag 60 ccctttc 67 298 67 DNA ArtificialSequence primer 298 tagagatggc gtatagttta tcgtactgct atcmnnatttatttcgccaa yccactccag 60 ccctttc 67 299 67 DNA Artificial Sequenceprimer 299 tagagatggc gtatagttta tcgtactgct mnntggattt atttcgccaayccactccag 60 ccctttc 67 300 67 DNA Artificial Sequence primer 300tagagatggc gtatagttta tcgtactmnn atctggattt atttcgccaa yccactccag 60ccctttc 67 301 67 DNA Artificial Sequence primer 301 tagagatggcgtatagttta tcgtmnngct atctggattt atttcgccaa yccactccag 60 ccctttc 67 30267 DNA Artificial Sequence primer 302 tagagatggc gtatagttta tmnnactgctatctggattt atttcgccaa yccactccag 60 ccctttc 67 303 67 DNA ArtificialSequence primer 303 tagagatggc gtatagttmn ncgtactgct atctggatttatttcgccaa yccactccag 60 ccctttc 67 304 67 DNA Artificial Sequenceprimer 304 cgttgtctct ggagatgrtg aatytatcct ttagagatgg cgtatamnntatcgtactgc 60 tatctgg 67 305 67 DNA Artificial Sequence primer 305cgttgtctct ggagatgrtg aatytatcct ttagagatgg cgtmnngttt atcgtactgc 60tatctgg 67 306 67 DNA Artificial Sequence primer 306 cgttgtctctggagatgrtg aatytatcct ttagagatgg mnnatagttt atcgtactgc 60 tatctgg 67 30767 DNA Artificial Sequence primer 307 cgttgtctct ggagatgrtg aatytatcctttagagamnn cgtatagttt atcgtactgc 60 tatctgg 67 308 67 DNA ArtificialSequence primer 308 cgttgtctct ggagatgrtg aatytatcct ttagmnntggcgtatagttt atcgtactgc 60 tatctgg 67 309 67 DNA Artificial Sequenceprimer 309 cgttgtctct ggagatgrtg aatytatcct tmnnagatgg cgtatagtttatcgtactgc 60 tatctgg 67 310 67 DNA Artificial Sequence primer 310cgttgtctct ggagatgrtg aatytatcmn ntagagatgg cgtatagttt atcgtactgc 60tatctgg 67 311 67 DNA Artificial Sequence primer 311 cgttgtctctggagatgrtg aatytmnnct ttagagatgg cgtatagttt atcgtactgc 60 tatctgg 67 31258 DNA Artificial Sequence primer 312 ataggtgttt ccattactat gtacaatgctctgactagam nngcaggaga tggaggcc 58 313 58 DNA Artificial Sequence primer313 ataggtgttt ccattactat gtacaatgct ctgactmnnc ctgcaggaga tggaggcc 58314 58 DNA Artificial Sequence primer 314 ataggtgttt ccattactatgtacaatgct ctgmnnagac ctgcaggaga tggaggcc 58 315 58 DNA ArtificialSequence primer 315 ataggtgttt ccattactat gtacaatgct mnnactagacctgcaggaga tggaggcc 58 316 58 DNA Artificial Sequence primer 316ataggtgttt ccattactat gtacaatmnn ctgactagac ctgcaggaga tggaggcc 58 31758 DNA Artificial Sequence primer 317 ataggtgttt ccattactat gtacmnngctctgactagac ctgcaggaga tggaggcc 58 318 58 DNA Artificial Sequence primer318 ataggtgttt ccattactat gmnnaatgct ctgactagac ctgcaggaga tggaggcc 58319 58 DNA Artificial Sequence primer 319 ataggtgttt ccattactmnntacaatgct ctgactagac ctgcaggaga tggaggcc 58 320 60 DNA ArtificialSequence primer 320 tggcttctgc aggtaccatt ccaaataggt gtttccattmnnatgtacaa tgctctgact 60 321 60 DNA Artificial Sequence primer 321tggcttctgc aggtaccatt ccaaataggt gtttccmnna ctatgtacaa tgctctgact 60 32260 DNA Artificial Sequence primer 322 tggcttctgc aggtaccatt ccaaataggtgttmnnatta ctatgtacaa tgctctgact 60 323 60 DNA Artificial Sequenceprimer 323 tggcttctgc aggtaccatt ccaaataggt mnntccatta ctatgtacaatgctctgact 60 324 60 DNA Artificial Sequence primer 324 tggcttctgcaggtaccatt ccaaatamnn gtttccatta ctatgtacaa tgctctgact 60 325 60 DNAArtificial Sequence primer 325 tggcttctgc aggtaccatt ccaamnnggtgtttccatta ctatgtacaa tgctctgact 60 326 60 DNA Artificial Sequenceprimer 326 tggcttctgc aggtaccatt cmnnataggt gtttccatta ctatgtacaatgctctgact 60 327 60 DNA Artificial Sequence primer 327 tggcttctgcaggtaccamn ncaaataggt gtttccatta ctatgtacaa tgctctgact 60 328 57 DNAArtificial Sequence primer 328 gaacctgtct gggactccag aaaaccggttggaaacmnna tagatcagga gctgtgg 57 329 57 DNA Artificial Sequence primer329 gaacctgtct gggactccag aaaaccggtt ggamnnttta tagatcagga gctgtgg 57330 57 DNA Artificial Sequence primer 330 gaacctgtct gggactccagaaaaccggtt mnnaacttta tagatcagga gctgtgg 57 331 57 DNA ArtificialSequence primer 331 gaacctgtct gggactccag aaaaccgmnn ggaaactttatagatcagga gctgtgg 57 332 57 DNA Artificial Sequence primer 332gaacctgtct gggactccag aaaamnngtt ggaaacttta tagatcagga gctgtgg 57 333 57DNA Artificial Sequence primer 333 gaacctgtct gggactccag amnnccggttggaaacttta tagatcagga gctgtgg 57 334 57 DNA Artificial Sequence primer334 gaacctgtct gggactccmn naaaccggtt ggaaacttta tagatcagga gctgtgg 57335 57 DNA Artificial Sequence primer 335 tgggggctga cggatccagcccacacccat tccagamnng ctgagtgaga acccaga 57 336 57 DNA ArtificialSequence primer 336 tgggggctga cggatccagc ccacacccat tccmnnagtgctgagtgaga acccaga 57 337 57 DNA Artificial Sequence primer 337tgggggctga cggatccagc ccacacccat mnnagaagtg ctgagtgaga acccaga 57 338 57DNA Artificial Sequence primer 338 tgggggctga cggatccagc ccacaccmnntccagaagtg ctgagtgaga acccaga 57 339 57 DNA Artificial Sequence primer339 tgggggctga cggatccagc ccacmnncat tccagaagtg ctgagtgaga acccaga 57340 57 DNA Artificial Sequence primer 340 tgggggctga cggatccagccmnnacccat tccagaagtg ctgagtgaga acccaga 57 341 57 DNA ArtificialSequence primer 341 tgggggctga cggatccamn ncacacccat tccagaagtgctgagtgaga acccaga 57 342 60 DNA Artificial Sequence primer 342cagagatggg ttgtagtatt tattgtcatc ccaccaaatm nntgcaagcc actccagggc 60 34360 DNA Artificial Sequence primer 343 cagagatggg ttgtagtatt tattgtcatcccaccamnng tctgcaagcc actccagggc 60 344 60 DNA Artificial Sequenceprimer 344 cagagatggg ttgtagtatt tattgtcatc ccamnnaatg tctgcaagccactccagggc 60 345 60 DNA Artificial Sequence primer 345 cagagatgggttgtagtatt tattgtcatc mnnccaaatg tctgcaagcc actccagggc 60 346 60 DNAArtificial Sequence primer 346 cagagatggg ttgtagtatt tattgtcmnnccaccaaatg tctgcaagcc actccagggc 60 347 60 DNA Artificial Sequenceprimer 347 cagagatggg ttgtagtatt tattmnnatc ccaccaaatg tctgcaagccactccagggc 60 348 60 DNA Artificial Sequence primer 348 cagagatgggttgtagtatt tmnngtcatc ccaccaaatg tctgcaagcc actccagggc 60 349 60 DNAArtificial Sequence primer 349 cagagatggg ttgtagtamn nattgtcatcccaccaaatg tctgcaagcc actccagggc 60 350 60 DNA Artificial Sequenceprimer 350 cttggagatg gtgagcctgc tcttcagaga tgggttgtam nntttattgtcatcccacca 60 351 60 DNA Artificial Sequence primer 351 cttggagatggtgagcctgc tcttcagaga tgggttmnng tatttattgt catcccacca 60 352 60 DNAArtificial Sequence primer 352 cttggagatg gtgagcctgc tcttcagagatggmnngtag tatttattgt catcccacca 60 353 60 DNA Artificial Sequenceprimer 353 cttggagatg gtgagcctgc tcttcagaga mnngttgtag tatttattgtcatcccacca 60 354 60 DNA Artificial Sequence primer 354 cttggagatggtgagcctgc tcttcagmnn tgggttgtag tatttattgt catcccacca 60 355 60 DNAArtificial Sequence primer 355 cttggagatg gtgagcctgc tcttmnnagatgggttgtag tatttattgt catcccacca 60 356 60 DNA Artificial Sequenceprimer 356 cttggagatg gtgagcctgc tmnncagaga tgggttgtag tatttattgtcatcccacca 60 357 60 DNA Artificial Sequence primer 357 cttggagatggtgagcctmn ncttcagaga tgggttgtag tatttattgt catcccacca 60 358 9 PRTArtificial Sequence synthetic antibody mutation 358 Phe Gln Ser Ser HisPhe Pro Trp Thr 1 5

What is claimed is:
 1. A grafted antibody, or functional fragmentthereof, comprising one or more complementarity determining regions(CDRs) having at least one amino acid substitution in one or more CDRsof a heavy chain CDR selected from the group consisting of SEQ IDNOS:26, 28 and 30 or a light chain CDR selected from the groupconsisting of SEQ ID NOS:20, 22 and 24, said grafted antibody orfunctional fragment thereof having specific binding activity for acryptic collagen epitope.
 2. An antibody, or functional fragmentthereof, comprising one or more CDRs selected from the group consistingof CDRs referenced as SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:54, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ IDNO:86, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, and SEQ IDNO:162, said antibody or functional fragment thereof having specificbinding activity for a cryptic collagen epitope.
 3. The antibody ofclaim 2, wherein said antibody, or functional fragment thereof,comprises a heavy chain CDR1 referenced as SEQ ID NO:26; a heavy chainCDR2 referenced as SEQ ID NO:28; a heavy chain CDR3 referenced as SEQ IDNO:63; a light chain CDR1 referenced as SEQ ID NO:20; a light chain CDR2referenced as SEQ ID NO:22; and a light chain CDR3 referenced as SEQ IDNO:77.
 4. The antibody of claim 2, wherein said antibody, or functionalfragment thereof, comprises a heavy chain CDR1 referenced as SEQ IDNO:26; a heavy chain CDR2 referenced as SEQ ID NO:28; a heavy chain CDR3referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:72; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77.
 5. The antibody of claim 2, whereinsaid antibody, or functional fragment thereof, comprises a heavy chainCDR1 referenced as SEQ ID NO:26; a heavy chain CDR2 referenced as SEQ IDNO:48; a heavy chain CDR3 referenced as SEQ ID NO:63; a light chain CDR1referenced as SEQ ID NO:20; a light chain CDR2 referenced as SEQ IDNO:22; and a light chain CDR3 referenced as SEQ ID NO:77.
 6. Theantibody of claim 2, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:45; aheavy chain CDR2 referenced as SEQ ID NO:154; a heavy chain CDR3referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:157; a light chain CDR2 referenced as SEQ ID NO:2 2; and a lightchain CDR3 referenced as SEQ ID NO:77.
 7. The antibody of claim 2,wherein said antibody, or functional fragment thereof, comprises a heavychain CDR1 referenced as SEQ ID NO:26; a heavy chain CDR2 referenced asSEQ ID NO:155; a heavy chain CDR3 referenced as SEQ ID NO:63; a lightchain CDR1 referenced as SEQ ID NO:158; a light chain CDR2 referenced asSEQ ID NO:22; and a light chain CDR3 referenced as SEQ ID NO:77.
 8. Theantibody of claim 2, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:46; aheavy chain CDR2 referenced as SEQ ID NO:155; a heavy chain CDR3referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:159; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77.
 9. The antibody of claim 2, whereinsaid antibody, or functional fragment thereof, comprises a heavy chainCDR1 referenced as SEQ ID NO:26; a heavy chain CDR2 referenced as SEQ IDNO:48; a heavy chain CDR3 referenced as SEQ ID NO:63; a light chain CDR1referenced as SEQ ID NO:160; a light chain CDR2 referenced as SEQ IDNO:22; and a light chain CDR3 referenced as SEQ ID NO:77.
 10. Theantibody of claim 2, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:45; aheavy chain CDR2 referenced as SEQ ID NO:155; a heavy chain CDR3referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:72; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77.
 11. The antibody of claim 2, whereinsaid antibody, or functional fragment thereof, comprises a heavy chainCDR1 referenced as SEQ ID NO:26; a heavy chain CDR2 referenced as SEQ IDNO:155; a heavy chain CDR3 referenced as SEQ ID NO:63; a light chainCDR1 referenced as SEQ ID NO:157; a light chain CDR2 referenced as SEQID NO:22; and a light chain CDR3 referenced as SEQ ID NO:77.
 12. Theantibody of claim 2, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:45; aheavy chain CDR2 referenced as SEQ ID NO:155; a heavy chain CDR3referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:160; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77.
 13. The antibody of claim 2, whereinsaid antibody, or functional fragment thereof, comprises a heavy chainCDR1 referenced as SEQ ID NO:46; a heavy chain CDR2 referenced as SEQ IDNO:155; a heavy chain CDR3 referenced as SEQ ID NO:63; a light chainCDR1 referenced as SEQ ID NO:160; a light chain CDR2 referenced as SEQID NO:22; and a light chain CDR3 referenced as SEQ ID NO:77.
 14. Theantibody of claim 2, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:45; aheavy chain CDR2 referenced as SEQ ID NO:162; a heavy chain CDR3referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:158; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77.
 15. The antibody of claim 2, whereinsaid antibody, or functional fragment thereof, comprises a heavy chainCDR1 referenced as SEQ ID NO:45; a heavy chain CDR2 referenced as SEQ IDNO:156; a heavy chain CDR3 referenced as SEQ ID NO:63; a light chainCDR1 referenced as SEQ ID NO:157; a light chain CDR2 referenced as SEQID NO:22; and a light chain CDR3 referenced as SEQ ID NO:77.
 16. Theantibody of claim 2, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:26; aheavy chain CDR2 referenced as SEQ ID NO:154; a heavy chain CDR3referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:157; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77.
 17. The antibody of claim 2, whereinsaid antibody, or functional fragment thereof, comprises a heavy chainCDR1 referenced as SEQ ID NO:45; a heavy chain CDR2 referenced as SEQ IDNO:155; a heavy chain CDR3 referenced as SEQ ID NO:63; a light chainCDR1 referenced as SEQ ID NO:157; a light chain CDR2 referenced as SEQID NO:22; and a light chain CDR3 referenced as SEQ ID NO:77.
 18. Theantibody of claim 2, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:46; aheavy chain CDR2 referenced as SEQ ID NO:154; a heavy chain CDR3referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:161; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77.
 19. The antibody of claim 2, whereinsaid antibody, or functional fragment thereof, comprises a heavy chainCDR1 referenced as SEQ ID NO:46; a heavy chain CDR2 referenced as SEQ IDNO:156; a heavy chain CDR3 referenced as SEQ ID NO:63; a light chainCDR1 referenced as SEQ ID NO:161; a light chain CDR2 referenced as SEQID NO:22; and a light chain CDR3 referenced as SEQ ID NO:77.
 20. Theantibody of claim 2, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:46; aheavy chain CDR2 referenced as SEQ ID NO:28; a heavy chain CDR3referenced as SEQ ID NO:63; a light chain CDR1 referenced as SEQ IDNO:20; a light chain CDR2 referenced as SEQ ID NO:22; and a light chainCDR3 referenced as SEQ ID NO:77.
 21. An antibody, or functional fragmentthereof, comprising a heavy chain polypeptide comprising one or moreCDRs having at least one amino acid substitution in one or more heavychain CDRs, said heavy chain CDRs selected from the group consisting ofa heavy chain CDR1 selected from the group consisting of CDRs referencedas SEQ ID NOS:26, 43, 44, 45, 46, and 47; a heavy chain CDR2 selectedfrom the group consisting of CDRs referenced as SEQ ID NOS:28, 48, 49,50, 51, 52, 53, 54, and 55; and a heavy chain CDR3 selected from thegroup consisting of CDRs referenced as SEQ ID NOS:30, 56, 57, 58, 59,60, 61, 62, 63, and 64, said antibody or functional fragment thereofhaving specific binding activity for a cryptic collagen epitope.
 22. Anantibody, or functional fragment thereof, comprising a light chainpolypeptide comprising one or more CDRs having at least one amino acidsubstitution in one or more light chain CDRs, said light chain CDRsselected from the group consisting of a light chain CDR1 selected fromthe group consisting of CDRs referenced as SEQ ID NOS:20, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, and 76; a light chain CDR2 referenced asSEQ ID NO:22:; and a light chain CDR3 selected from the group consistingof CDRs referenced as SEQ ID NOS:24, 77, 78, 79, 80, 81, 82, 83, 84, 85,and 86, said antibody or functional fragment thereof having specificbinding activity for a cryptic collagen epitope.
 23. A grafted antibody,or functional fragment thereof, comprising one or more complementaritydetermining regions (CDRs) having at least one amino acid substitutionin one or more CDRs of a heavy chain CDR selected from the groupconsisting of SEQ ID NOS:38, 40 and 42 or a light chain CDR selectedfrom the group consisting of SEQ ID NOS:32, 34 and 36, said graftedantibody or functional fragment thereof having specific binding activityfor a cryptic collagen epitope.
 24. An antibody, or functional fragmentthereof, comprising one or more CDRs selected from the group consistingof CDRs referenced as SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ IDNO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ IDNO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ IDNO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109,SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ IDNO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQID NO:11 9, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123,SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ IDNO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137,SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ IDNO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151,SEQ ID NO:152, SEQ ID NO:153 and SEQ ID NO:358, said antibody orfunctional fragment thereof having specific binding activity for acryptic collagen epitope.
 25. The antibody of claim 24, wherein saidantibody, or functional fragment thereof, comprises a heavy chain CDR1referenced as SEQ ID NO:38; a heavy chain CDR2 referenced as SEQ IDNO:40; a heavy chain CDR3 referenced as SEQ ID NO:103; a light chainCDR1 referenced as SEQ ID NO:32; a light chain CDR2 referenced as SEQ IDNO:34; and a light chain CDR3 referenced as SEQ ID NO:36.
 26. Theantibody of claim 24, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:38; aheavy chain CDR2 referenced as SEQ ID NO:92; a heavy chain CDR3referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQ IDNO:32; a light chain CDR2 referenced as SEQ ID NO:34; and a light chainCDR3 referenced as SEQ ID NO:130.
 27. The antibody of claim 24, whereinsaid antibody, or functional fragment thereof, comprises a heavy chainCDR1 referenced as SEQ ID NO:147; a heavy chain CDR2 referenced as SEQID NO:92; a heavy chain CDR3 referenced as SEQ ID NO:103; a light chainCDR1 referenced as SEQ ID NO:149; a light chain CDR2 referenced as SEQID NO:34; and a light chain CDR3 referenced as SEQ ID NO:130.
 28. Theantibody of claim 24, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:147; aheavy chain CDR2 referenced as SEQ ID NO:92; a heavy chain CDR3referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQ IDNO:150; a light chain CDR2 referenced as SEQ ID NO:34; and a light chainCDR3 referenced as SEQ ID NO:130.
 29. The antibody of claim 24, whereinsaid antibody, or functional fragment thereof, comprises a heavy chainCDR1 referenced as SEQ ID NO:147; a heavy chain CDR2 referenced as SEQID NO:93; a heavy chain CDR3 referenced as SEQ ID NO:103; a light chainCDR1 referenced as SEQ ID NO:149; a light chain CDR2 referenced as SEQID NO:34; and a light chain CDR3 referenced as SEQ ID NO:130.
 30. Theantibody of claim 24, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:147; aheavy chain CDR2 referenced as SEQ ID NO:144; a heavy chain CDR3referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQ IDNO:149; a light chain CDR2 referenced as SEQ ID NO:34; and a light chainCDR3 referenced as SEQ ID NO:130.
 31. The antibody of claim 24, whereinsaid antibody, or functional fragment thereof, comprises a heavy chainCDR1 referenced as SEQ ID NO:147; a heavy chain CDR2 referenced as SEQID NO:93; a heavy chain CDR3 referenced as SEQ ID NO:10 3; a light chainCDR1 referenced as SEQ ID NO:151; a light chain CDR2 referenced as SEQID NO:34; and a light chain CDR3 referenced as SEQ ID NO:130.
 32. Theantibody of claim 24, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:147; aheavy chain CDR2 referenced as SEQ ID NO:92; a heavy chain CDR3referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQ IDNO:151; a light chain CDR2 referenced as SEQ ID NO:34; and a light chainCDR3 referenced as SEQ ID NO:130.
 33. The antibody of claim 24, whereinsaid antibody, or functional fragment thereof, comprises a heavy chainCDR1 referenced as SEQ ID NO:147; a heavy chain CDR2 referenced as SEQID NO:93; a heavy chain CDR3 referenced as SEQ ID NO:103; a light chainCDR1 referenced as SEQ ID NO:152; a light chain CDR2 referenced as SEQID NO:34; and a light chain CDR3 referenced as SEQ ID NO:358.
 34. Theantibody of claim 24, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:148; aheavy chain CDR2 referenced as SEQ ID NO:93; a heavy chain CDR3referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQ IDNO:150; a light chain CDR2 referenced as SEQ ID NO:34; and a light chainCDR3 referenced as SEQ ID NO:130.
 35. The antibody of claim 24, whereinsaid antibody, or functional fragment thereof, comprises a heavy chainCDR1 referenced as SEQ ID NO:147; a heavy chain CDR2 referenced as SEQID NO:93; a heavy chain CDR3 referenced as SEQ ID NO:103; a light chainCDR1 referenced as SEQ ID NO:115; a light chain CDR2 referenced as SEQID NO:34; and a light chain CDR3 referenced as SEQ ID NO:130.
 36. Theantibody of claim 24, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:147; aheavy chain CDR2 referenced as SEQ ID NO:40; a heavy chain CDR3referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQ IDNO:153; a light chain CDR2 referenced as SEQ ID NO:34; and a light chainCDR3 referenced as SEQ ID NO:130.
 37. The antibody of claim 24, whereinsaid antibody, or functional fragment thereof, comprises a heavy chainCDR1 referenced as SEQ ID NO:147; a heavy chain CDR2 referenced as SEQID NO:92; a heavy chain CDR3 referenced as SEQ ID NO:103; a light chainCDR1 referenced as SEQ ID NO:116; a light chain CDR2 referenced as SEQID NO:34; and a light chain CDR3 referenced as SEQ ID NO:130.
 38. Theantibody of claim 24, wherein said antibody, or functional fragmentthereof, comprises a heavy chain CDR1 referenced as SEQ ID NO:147; aheavy chain CDR2 referenced as SEQ ID NO:93; a heavy chain CDR3referenced as SEQ ID NO:103; a light chain CDR1 referenced as SEQ IDNO:116; a light chain CDR2 referenced as SEQ ID NO:34; and a light chainCDR3 referenced as SEQ ID NO:130.
 39. The antibody of claim 24, whereinsaid antibody, or functional fragment thereof, comprises a heavy chainCDR1 referenced as SEQ ID NO:38; a heavy chain CDR2 referenced as SEQ IDNO:93; a heavy chain CDR3 referenced as SEQ ID NO:103; a light chainCDR1 referenced as SEQ ID NO:32; a light chain CDR2 referenced as SEQ IDNO:34; and a light chain CDR3 referenced as SEQ ID NO:130.
 40. Anantibody, or functional fragment thereof, comprising a heavy chainpolypeptide comprising one or more CDRs having at least one amino acidsubstitution in one or more heavy chain CDRs, said heavy chain CDRsselected from the group consisting of a heavy chain CDR1 selected fromthe group consisting of CDRs referenced as SEQ ID NOS:38, 87, 88, 89,90, 91, 147 and 148; a heavy chain CDR2 selected from the groupconsisting of CDRs referenced as SEQ ID NOS:40, 92, 93, 94, 95 and 144;and a heavy chain CDR3 selected from the group consisting of CDRsreferenced as SEQ ID NOS:42, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108 and 109, said antibody or functional fragment thereofhaving specific binding activity for a cryptic collagen epitope.
 41. Anantibody, or functional fragment thereof, comprising a light chainpolypeptide comprising one or more CDRs having at least one amino acidsubstitution in one or more light chain CDRs, said light chain CDRsselected from the group consisting of a light chain CDR1 selected fromthe group consisting of CDRs referenced as SEQ ID NOS:32, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 146, 149, 150, 151, 152 and 153; alight chain CDR2 referenced as SEQ ID NOS:34, 120, 121, 122, 123, 124and 125; and a light chain CDR3 selected from the group consisting ofCDRs referenced as SEQ ID NOS:36, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 145 and 358, saidantibody or functional fragment thereof having specific binding activityfor a cryptic collagen epitope.
 42. The grafted antibody of any ofclaims 1-41, wherein said functional fragment is selected from the groupconsisting of Fv, Fab, F(ab)₂ and scFV.
 43. A nucleic acid encoding theantibody of any of claims 1-41.
 44. A method of targeting angiogenicvasculature, comprising administering an antibody, or functionalfragment thereof, said antibody or functional fragment thereofcomprising one or more complementarity determining regions (CDRs) havingat least one amino acid substitution in one or more CDRs of a heavychain CDR selected from the group consisting of SEQ ID NOS:26, 28 and 30or a light chain CDR selected from the group consisting of SEQ IDNOS:20, 22 and 24, and said antibody or functional fragment thereofhaving specific binding activity for a cryptic collagen epitope.
 45. Themethod of claim 44, wherein said antibody or functional fragmentcomprises one or more CDRs selected from the group consisting of CDRsreferenced as SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56,SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61,SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81,SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86,SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ IDNO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, and SEQ ID NO:162.46. The method of claim 44, wherein said antibody, or functionalfragment thereof, further comprises a therapeutic moiety.
 47. The methodof claim 44, wherein said antibody, or functional fragment thereof,further comprises a detectable moiety.
 48. A method of inhibitingangiogenesis, comprising administering an antibody, or functionalfragment thereof, said antibody or functional fragment thereofcomprising one or more complementarity determining regions (CDRs) havingat least one amino acid substitution in one or more CDRs of a heavychain CDR selected from the group consisting of SEQ ID NOS:26, 28 and 30or a light chain CDR selected from the group consisting of SEQ IDNOS:20, 22 and 24, and said antibody or functional fragment thereofhaving specific binding activity for a cryptic collagen epitope.
 49. Themethod of claim 48, wherein said antibody or functional fragmentcomprises one or more CDRs selected from the group consisting of CDRsreferenced as SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56,SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61,SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81,SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86,SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ IDNO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, and SEQ ID NO:162.50. The method of claim 48, wherein said antibody, or functionalfragment thereof, further comprises a therapeutic moiety.
 51. A methodof targeting a tumor, comprising administering an antibody, orfunctional fragment thereof, said antibody or functional fragmentthereof comprising one or more complementarity determining regions(CDRs) having at least one amino acid substitution in one or more CDRsof a heavy chain CDR selected from the group consisting of SEQ IDNOS:26, 28 and 30 or a light chain CDR selected from the groupconsisting of SEQ ID NOS:20, 22 and 24, and said antibody or functionalfragment thereof having specific binding activity for a cryptic collagenepitope.
 52. The method of claim 51, wherein said antibody or functionalfragment comprises one or more CDRs selected from the group consistingof CDRs referenced as SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ IDNO:86, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, and SEQ IDNO:162.
 53. The method of claim 51, wherein said antibody, or functionalfragment thereof, further comprises a therapeutic moiety.
 54. The methodof claim 51, wherein said antibody, or functional fragment thereof,further comprises a detectable moiety.
 55. A method of inhibiting tumorgrowth, comprising administering an antibody, or functional fragmentthereof, said antibody or functional fragment thereof comprising one ormore complementarity determining regions (CDRs) having at least oneamino acid substitution in one or more CDRs of a heavy chain CDRselected from the group consisting of SEQ ID NOS:26, 28 and 30 or alight chain CDR selected from the group consisting of SEQ ID NOS:20, 22and 24, and said antibody or functional fragment thereof having specificbinding activity for a cryptic collagen epitope.
 56. The method of claim55, wherein said antibody or functional fragment comprises one or moreCDRs selected from the group consisting of CDRs referenced as SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ IDNO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ IDNO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:154, SEQ IDNO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQID NO:160, SEQ ID NO:161, and SEQ ID NO:162.
 57. The method of claim 55,wherein said antibody, or functional fragment thereof, further comprisesa therapeutic moiety.
 58. A method of detecting angiogenic vasculature,comprising contacting angiogenic vasculature with an antibody, orfunctional fragment thereof, said antibody or functional fragmentthereof comprising one or more complementarity determining regions(CDRs) having at least one amino acid substitution in one or more CDRsof a heavy chain CDR selected from the group consisting of SEQ IDNOS:26, 28 and 30 or a light chain CDR selected from the groupconsisting of SEQ ID NOS:20, 22 and 24, and said antibody or functionalfragment thereof having specific binding activity for a cryptic collagenepitope.
 59. The method of claim 58, wherein said antibody or functionalfragment comprises one or more CDRs selected from the group consistingof CDRs referenced as SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ IDNO:86, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, and SEQ IDNO:162.
 60. The method of claim 58, wherein said antibody, or functionalfragment thereof, further comprises a detectable moiety.
 61. A method ofinhibiting metastasis, comprising administering an antibody, orfunctional fragment thereof, said antibody or functional fragmentthereof comprising one or more complementarity determining regions(CDRs) having at least one amino acid substitution in one or more CDRsof a heavy chain CDR selected from the group consisting of SEQ IDNOS:26, 28 and 30 or a light chain CDR selected from the groupconsisting of SEQ ID NOS:20, 22 and 24, and said antibody or functionalfragment thereof having specific binding activity for a cryptic collagenepitope.
 62. The method of claim 61, wherein said antibody or functionalfragment comprises one or more CDRs selected from the group consistingof CDRs referenced as SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ IDNO:86, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, and SEQ IDNO:162.
 63. The method of claim 61, wherein said antibody, or functionalfragment thereof, further comprises a therapeutic moiety.
 64. A methodof targeting angiogenic vasculature, comprising administering anantibody, or functional fragment thereof, said antibody or functionalfragment thereof comprising one or more complementarity determiningregions (CDRs) having at least one amino acid substitution in one ormore CDRs of a heavy chain CDR selected from the group consisting of SEQID NOS:38, 40 and 42 or a light chain CDR selected from the groupconsisting of SEQ ID NOS:32, 34 and 36, said grafted antibody orfunctional fragment thereof having specific binding activity for acryptic collagen epitope.
 65. The method of claim 64, wherein saidantibody or functional fragment comprises one or more CDRs selected fromthe group consisting of CDRs referenced as SEQ ID NO:87, SEQ ID NO:88,SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93,SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98,SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ IDNO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112,SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ IDNO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126,SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ IDNO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140,SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ IDNO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153 and SEQ IDNO:358.
 66. The method of claim 64, wherein said antibody, or functionalfragment thereof, further comprises a therapeutic moiety.
 67. The methodof claim 64, wherein said antibody, or functional fragment thereof,further comprises a detectable moiety.
 68. A method of inhibitingangiogenesis, comprising administering an antibody, or functionalfragment thereof, said antibody or functional fragment thereofcomprising one or more complementarity determining regions (CDRs) havingat least one amino acid substitution in one or more CDRs of a heavychain CDR selected from the group consisting of SEQ ID NOS:38, 40 and 42or a light chain CDR selected from the group consisting of SEQ IDNOS:32, 34 and 36, said grafted antibody or functional fragment thereofhaving specific binding activity for a cryptic collagen epitope.
 69. Themethod of claim 68, wherein said antibody or functional fragmentcomprises one or more CDRs selected from the group consisting of CDRsreferenced as SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90,SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95,SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100,SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ IDNO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114,SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:11 8, SEQ IDNO:11 9, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128,SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ IDNO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142,SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ IDNO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQID NO:152, SEQ ID NO:153 and SEQ ID NO:358.
 70. The method of claim 68,wherein said antibody, or functional fragment thereof, further comprisesa therapeutic moiety.
 71. A method of targeting a tumor, comprisingadministering an antibody, or functional fragment thereof, said antibodyor functional fragment thereof comprising one or more complementaritydetermining regions (CDRs) having at least one amino acid substitutionin one or more CDRs of a heavy chain CDR selected from the groupconsisting of SEQ ID NOS:38, 40 and 42 or a light chain CDR selectedfrom the group consisting of SEQ ID NOS:32, 34 and 36, said graftedantibody or functional fragment thereof having specific binding activityfor a cryptic collagen epitope.
 72. The method of claim 71, wherein saidantibody or functional fragment comprises one or more CDRs selected fromthe group consisting of CDRs referenced as SEQ ID NO:87, SEQ ID NO:88,SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93,SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98,SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ IDNO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112,SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ IDNO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126,SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ IDNO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140,SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ IDNO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQID NO:150, SEQ ID NO:151, SEQ ID NO:15 2, SEQ ID NO:153 and SEQ IDNO:358.
 73. The method of claim 71, wherein said antibody, or functionalfragment thereof, further comprises a therapeutic moiety.
 74. The methodof claim 71, wherein said antibody, or functional fragment thereof,further comprises a detectable moiety.
 75. A method of inhibiting tumorgrowth, comprising administering an antibody, or functional fragmentthereof, said antibody or functional fragment thereof comprising one ormore complementarity determining regions (CDRs) having at least oneamino acid substitution in one or more CDRs of a heavy chain CDRselected from the group consisting of SEQ ID NOS:38, 40 and 42 or alight chain CDR selected from the group consisting of SEQ ID NOS:32, 34and 36, said grafted antibody or functional fragment thereof havingspecific binding activity for a cryptic collagen epitope.
 76. The methodof claim 75, wherein said antibody or functional fragment comprises oneor more CDRs selected from the group consisting of CDRs referenced asSEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91,SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96,SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ IDNO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115,SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ IDNO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129,SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ IDNO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ IDNO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQID NO:153 and SEQ ID NO:358.
 77. The method of claim 75, wherein saidantibody, or functional fragment thereof, further comprises atherapeutic moiety.
 78. A method of detecting angiogenic vasculature,comprising contacting angiogenic vasculature with an antibody, orfunctional fragment thereof, said antibody or functional fragmentthereof comprising one or more complementarity determining regions(CDRs) having at least one amino acid substitution in one or more CDRsof a heavy chain CDR selected from the group consisting of SEQ IDNOS:38, 40 and 42 or a light chain CDR selected from the groupconsisting of SEQ ID NOS:32, 34 and 36, said grafted antibody orfunctional fragment thereof having specific binding activity for acryptic collagen epitope.
 79. The method of claim 78, wherein saidantibody or functional fragment comprises one or more CDRs selected fromthe group consisting of CDRs referenced as SEQ ID NO:87, SEQ ID NO:88,SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93,SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98,SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ IDNO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112,SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ IDNO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126,SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ IDNO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140,SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ IDNO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153 and SEQ IDNO:358.
 80. The method of claim 78, wherein said antibody, or functionalfragment thereof, further comprises a detectable moiety.
 81. A method ofinhibiting tumor growth, comprising administering an antibody, orfunctional fragment thereof, said antibody or functional fragmentthereof comprising one or more complementarity determining regions(CDRs) having at least one amino acid substitution in one or more CDRsof a heavy chain CDR selected from the group consisting of SEQ IDNOS:38, 40 and 42 or a light chain CDR selected from the groupconsisting of SEQ ID NOS:32, 34 and 36, said grafted antibody orfunctional fragment thereof having specific binding activity for acryptic collagen epitope.
 82. The method of claim 81, wherein saidantibody or functional fragment comprises one or more CDRs selected fromthe group consisting of CDRs referenced as SEQ ID NO:87, SEQ ID NO:88,SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93,SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98,SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ IDNO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112,SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ IDNO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126,SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ IDNO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140,SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ IDNO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153 and SEQ IDNO:358.
 83. The method of claim 81, wherein said antibody, or functionalfragment thereof, further comprises a therapeutic moiety.